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Research ArticleExtraction and Characterization of Organ Components ofthe Malaysian Sea Cucumber Holothuria leucospilota YieldedBioactives Exhibiting Diverse Properties

Abdoulie Ceesay1 Mariana Nor Shamsudin1

Mohammed Aliyu-Paiko 12 Intan Safinar Ismail 3

Muhammad Farhan Nazarudin 1 and Norfarrah Mohamed Alipiah1

1Laboratory of Marine Biotechnology Institute of Bioscience Universiti Putra Malaysia 43400 UPM Serdang Selangor Malaysia2Biochemistry Department Faculty of Natural Sciences Ibrahim Badamasi Babangida (IBB) University P M B 11 Lapai Nigeria3Chemistry Department Faculty of Science Universiti Putra Malaysia 43400 UPM Serdang Selangor Malaysia

Correspondence should be addressed to Muhammad Farhan Nazarudin m farhannazaupmedumy

Received 19 January 2019 Accepted 5 March 2019 Published 15 April 2019

Academic Editor Jane Hanrahan

Copyright copy 2019 Abdoulie Ceesay et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of the present study was to extract and characterize bioactive components from separate body organs of Holothurialeucospilota Preliminary qualitative assessment of the crude extractswaspositive for phenols terpenoids carbohydrates flavonoidssaponins glycosides cardiac glycosides steroids phlobatannins and tannins in all body organs evaluated Phenolics were the mostabundant group of bioactives accounting for approximately 80 The extraction solvent mixtures that yielded most compoundsevaluated were methanolacetone (31 vv) and methanoldistilled water (31 vv) In other analyses GC-MS data revealeddiverse metabolic and biologically active compounds where those in high concentrations included 2-Pentanone 4-hydroxy-4-methyl- among the ketones phenol- 24-bis(11-dimethylethyl)- a phenol group and 2-Chlorooctane a hydrocarbon Among FAand their methylethyl esters n-hexadecanoic acid 581114-eicosatetraenoic acid ethyl ester (arachidonic acid) and 58111417-eicosapentaenoic acid methyl ester (EPA) were among the most abundant FAMEs accounting for approximately 50 of thesubgroups measured Data from GC-FID analysis revealed methyl laurate (C120) methyl myristate (C140) methyl palmitate(C160) and methyl stearate (180) methyl esters as the most abundant saturated FA whereas cis-9-oleic methyl ester (C181)and methyl linoleate (C182) were found as the major monounsaturated FA and PUFA FAMEs respectively in the body wallof the species Taken together the extraction and characterization of different categories of metabolically and biologically activecompounds in various organ extracts of H leucospilota suggest that the species is potentially a rich source of cholesterol-loweringantioxidant antimicrobial and anticancer agents These substances are known to benefit human health and assist in diseasepreventionThese findings justify the use of sea cucumbers in traditional folklore medication and the current interest and attentionfocused on the species to mine for bioactives in new drugs research

1 Introduction

Sea cucumbers are high value marine organisms that arefound well distributed worldwide Expert estimate suggeststhat there are presently about 1 400 species of sea cucumbersand new species continue to be identified and classifiedamong six valid orders Apodida Aspidochirotida Elasipo-didaMolpadiidaDendrochirotida andDactylochirotida [1]For centuries different species of sea cucumbers have been

fished primarily for food [2] whereas products of biomedicalimportance from them have also been used locally andinternationally in traditional medications [3]

As food many species of edible sea cucumbers have highnutritional value because of their content of high-qualityproteins which comprise a rich profile of amino acids and lowlevel of fat that is supported by a rich combination of desirabletrace minerals In summary the species is highly regarded inAsia as a rare traditional delicacy an important traditional

HindawiBioMed Research InternationalVolume 2019 Article ID 2640684 16 pageshttpsdoiorg10115520192640684

2 BioMed Research International

medication and aphrodisiac dating back many centuries [4]To highlight the historical importance of these organisms inthe Far East [5] opined that sea cucumbers have been usefulin folk medicine where data in the literature suggests thatabout fifty-two species are commercially exploited as foodand most of these are native to the tropics and subtropics [6]

In addition to consumption as functional food resultsof published studies also suggest that various organs of seacucumbers contain numerous bioactives that show biologicalactivity which may have potential as therapeutic agentsin human medicine and for veterinary applications Someimportant compounds already identified in these marinespecies include sulfated polysaccharides such as fucosylatedchondroitin sulfate [7] sterols sterol glycosides frondogeninand its glycosides [8 9] Furthermore lectins heparincerebrosides gangliosides omega-6 and omega-3 fatty acids(n-6 and n-3 FA) that are known to exhibit multiple biologicalactivities in living cells are also among the substances isolatedfrom sea cucumbers These compounds are now in use inanticancer antiangiogenic anticoagulant antihypertensiveanti-inflammatory antimicrobial antifungal antioxidantantithrombotic and antitumor applications [8]

Among the species of sea cucumber so far evaluatedHolothuria leucospilota is particularly widely distributed inthe shallow reefs of tropical subtropical and Indo-Pacificregions including the Red Sea [10ndash12] They are also foundliving in the Pacific Ocean from Northern Australia south-wards to LordHowe and have been discovered in the tropicalwaters of the Indo-West Pacific feeding on phytoplanktonmicro- and macroalgae and bacteria [13] Analysis of Hleucospilota has revealed some unique additional bioactivecompounds that are of therapeutic significance includingphenolic compounds [14] essential and free fatty acids [1516] gangliosides [17] and carotenoids [18] among manyothers In other words the detection of these compounds inthe species has been considered as strong evidence to supportthe use and application of extracts from H Leucospilota infolklore medicine to treat several infections and diseases

Despite this relative advantage majority of studiesencountered on the species in the literature are primarilyfocused on its content of triterpene glycosides or some otherspecific compounds of interest to researchers The completeanalysis of bioactives in the species is yet to be carried outsuggesting that more quantitative research is required toevaluate the entire body organs ofH leucospilota for bioactivecomponents The present study was therefore aimed at fullyextracting and characterizing the complete bioactive com-ponents in the body organs of adult H leucospilota sampledfrom the coastal shores ofMalaysia in order to determine themetabolically and biologically active substances and the fattyacid (FA) constituents

2 Materials and Methods

21 Source and Preparation of Sea Cucumber Samples Seacucumbers used in the present study were sampled fromTeluk Kemang near Port Dickson (2∘ 271015840 466110158401015840N 101∘501015840 429810158401015840E) Malaysia Live adult samples were collectedby hand from the sea shores in plastic sample containers

during low tides Samples were washed free of debris withclean sea water kept in transparent polyethylene bags inchilled sampling boxes and transported to the laboratoryfacilities of Marine Biotechnology Institute of Biosciences(IBS) Universiti Putra Malaysia (UPM) Serdang SelangorMalaysia

In the laboratory liveH leucospilota samples were furthercleaned of sand salt water and left-over debris with distilledwater and then cut into small pieces (2 cm times 2 cm) withsterile knives forceps and other dissecting tools The bodywall Cuvierian tubules and visceral organs were separatedwashed and kept in separate sample bottles Separated organsamples were then lyophilized using a bench top K-manifoldfreeze-dryer (FreeZone Labconco USA) for 120 hours Thefreeze-dried samples were then ground with a blender (MagicBullet Model MBR-1101G USA) to fine powder quantifiedon electronic weighing balance (Model B12-FAJA Taiwan)and kept in dry sample bottles The dry powdered samples inbottles were covered with aluminum foil papers and storedat -20∘C until used during subsequent analyses within threeweeksThe powdered samples were appropriately labeled andidentified as body wall (BW) Cuvierian tubules (CT) andvisceral organs (VO) in subsequent evaluations

22 Crude Sample Extraction Procedure Extraction of bioac-tive constituents was performed ondry powderedH leucospi-lota organ samples using Soxhlet and conventional solventextraction techniques Briefly during the Soxhlet extractionknown volumes (vv label) of analytical grade solventsmethanolacetone (31 MA) isopropanolacetone (31 PA)ethanoldistilled water (31 ED) methanoldistilled water(31 MD) and iso-propanoldistilled water (31 PD) wereused Repeated extraction was carried out on each separatelyweighed sample per solvent mixture to extract bioactiveconstituents from various powdered sea cucumber organsamples Each sample extraction procedure lasted about 5hours Subsequently fractions from each repeated solventextraction were pooled together and filtered with Whatman(No 1) filter paper and the filtrate was evaporated to drynesson a rotary evaporator

In the conventional solvent extraction procedure thepowdered organ samples were weighed and dissolved in ana-lytical grade (vv label) acetone (A) and methanolacetone(11 MA) Approximately 200 g of BW 70 g CT and 73 gVO were extracted in 2 L 700 mL and 730 mL of extractionsolvent respectively (representing a concentration of 1 g oforgan sample in 10 mL of extraction solvent) Extractionwas carried out under ambient temperature for 4 days withconstant shaking according to slight modification to themethod described by [20] All extractions were carried outunder aseptic conditions in clean dry beakersThe procedurewas repeated many times (between twelve to fifteen cycles)until no further color changes were observed in the solventsBatches of the extracts for each sample were then pooledand filtered and the filtrate was concentrated on a rotaryevaporator as previously described Water (moisture) wasremoved from the extracts by freeze-drying and the dry organextracts (BW CT and VO) were then used in subsequentevaluations immediately or stored at -20∘C until used

BioMed Research International 3

23 Qualitative Characterization of Bioactives in H leucospi-lota Crude Organ Extracts The screening procedure usedto characterize the bioactives in crude organ extracts of Hleucospilota was carried out according to slight modificationto that adopted by [21] In this analyticalmethod the presenceof bioactive compounds that contribute to flavor color andother important characteristics in the organs of sea cucumberwas determined

To identify groups of compounds in the crude organextracts however standard chemical screening was per-formed according to the procedure published by otherauthors [22ndash25] Briefly flavonoidswere screened using FeCl3via Pewrsquos and Shinodarsquos tests glycosides were determined byKeller- Killani test saponins were evaluated by frothing testtannins were evaluated by FeCl3 and lead acetate terpenoidswere evaluated by Salkowskirsquos test carbohydrates were deter-mined following Barfoedrsquos Fehlingrsquos and Molischrsquos tests andsteroids were evaluated by Liebermann-Burchard test Thetests for phenolics phlobatannins and cardiac glycosideswere carried out in accordance with the procedure reportedin the literature [22ndash25]

24 Quantitative Characterization for Bioactive Groups inH leucospilota Organ Extracts

241 Determination of Total Phenolics Content Total pheno-lics content (TPC) was determined using the Folin-Ciocalteureagent according to slight modification of the methodpublished by [26] To determine TPC 05 mL of dry BWcrude extract (50 mgmL) was added to 25 mL of Folin-Ciocalteu reagent diluted with distilled water (110 vv) and125 mL of 20 aqueous sodium carbonate in a test tubecovered with aluminum foil The test tube was vortex-mixedand incubated at ambient temperature (28∘C) for 40minutesThe absorbance of the mixture was then measured at 725 nmin a spectrophotometer (Shimadzu UV-1601 UV-VIS visibleJapan) against a blank containing the same mixture exceptthat Folin-Ciocalteu reagent was replacedwith distilledwaterTPC for the extract was calculated from an equation obtainedfrom a calibration curve generated with solution of gallic aciddissolved inmethanol and expressed as gallic acid equivalents(GAE) per gram of extract The content of phenolics in gallicacid equivalent was calculated using the following formula

C = (C times V)m

(1)

where C is the total content of phenolic compoundsmgg dryextract in GAE c is the concentration of gallic acid equivalentfrom the calibration curve 120583gmL V is the volume of extractused mL and m is the dry weight of extract g

242 Determination of Total Content of Flavonoids Totalflavonoids content (TFC) was determined using aluminumchloride colorimetric assay following slight modificationsto the procedure reported by [27] TFC was carried out byadding 05 mL of the BW crude extract (50 mgmL) to 03mL of NaNO2 (5) in a test tube covered with aluminum foiland mixing After incubation for 5 minutes 03 mL of AlCl3

(10) was added into the test tube and further incubatedfor 1 minute Approximately 2 mL of NaOH (1M) and 14mL of distilled water were added to the mixture and vortex-mixed before the absorbance was measured at 510 nm in aspectrophotometer TFC for each extract was calculated fromthe equation obtained from a calibration curve generatedwith a solution of rutin dissolved in methanol where TFCwas expressed as rutin equivalents (RE) per gram of extractThe content of flavonoids in rutin equivalent was calculatedby the following formula

C = (C times V)m

(2)

where C is the total content of flavonoid compounds mggdry extract in RE c is the concentration of rutin equivalentestablished from the calibration curve 120583gmL V is thevolumeof extract usedmL andm is the dryweight of extractg

243 Determination of Total Content of Saponins Totalsaponins content (TSC) was determined according to minormodifications to the vanillin-sulphuric acid calorimetricmethod as reported by [28] To determine TSC 025 mL ofthe BW crude extract (50 mgmL) was added to 025 mL ofvanillin reagent (8) and 025mL of 72H2SO4 in a test tubecovered with aluminum foil The test tube was vortex-mixedand warmed on a hot water bath maintained at 60∘C After10 minutes the sample mixture was cooled in ice cold waterfor 4minutes and the absorbance was read at 544 nm using aspectrophotometer TSC for each extract was calculated fromthe equation obtained from a calibration curve generatedwith a solution of diosgenin dissolved in methanol and theTSC was expressed as diosgenin equivalents (DE) per gramof extract The content of saponins in diosgenin equivalentwas calculated by the following formula

C = (C times V)m

(3)

where C is the total content of saponin compounds mgg dryextract in DE c is the concentration of diosgenin establishedfrom the calibration curve 120583gmL V is the volume of extractused mL and m is the dry weight of extract g

25 GC-MS Analysis to Characterize and Quantify Bioac-tives in H leucospilota Extracts Freeze-dried sea cucumberorgan extracts were characterized quantitatively via GasChromatography-Mass Spectrometry (GC-MS) according toslight modifications to the method adopted by [29] usinga Shimadzu QP5050A GC-MS system In the experimentalprocedure 10 120583L of sample was separated on a Zebron (ZB-FFAP GC-17A version 3 USA) column (30 m times 025 mm)Splitless injection was performed using a purge time of 1minute Helium represented the carrier gas at a flow rate of1mLmin The column temperature was maintained at 50∘Cfor 3 minutes then programmed at 250∘C for 10 minutesand maintained at 250∘C for 30 minutes The inlet and thedetector temperatures were set at 250∘C and the solvent cuttime was set at 450 minutes Identification of peaks was


Table 1 Qualitative biochemical analysis of crude extracts of different organ samples of H leucospilota in various solvents

Compound Groups Organs in Solvent ExtractsACT MACT AVO MAVO ABW MABW PABW EDBW MDBW PDBW

Phenols + + + + + + + + + +

Terpenes + + + + + + + + + +

Carbohydrates + + + + + + + + + +

Phlobatannins - - - - + + - - - -Tannins - - + + + + + + + +

Steroids - - + - + + + + +

Saponins - + - + - + + + + +

Cardiac glycosides - + - + - + + + + +

Glycosides - + - + - + + + + +

Flavonoids - - - + + + + + + +

lowast(+)Present( -)Absent ACT (acetoneCuvierian tubule) MACT (methanol-acetoneCuvierian tubule) AVO (acetonevisceral organ) MAVO (methanol-acetonevisceral organ) ABW (acetonebody wall) MABW (methanol-acetonebody wall) PABW (isopropanol-acetonebody wall) EDBW (ethanol-distilled waterbody wall) MDBW (methanol-distilled waterbody wall) and PDBW (isopropanol-distilled waterbody wall)[19]

based on a computer-based program matching the massspectra with those in the library for National Institute ofStandards and Technology (NlST 08 and NIST 08s) This wasdone by comparing retention time data with that obtainedfor authentic laboratory standards Individual detected peakareas were quantified and expressed as percentage of totalcomponents detected

26 Quantitative Fatty Acids Analysis via GC-FID Extractedlipids from dry BW samples were esterified to form fatty acidmethyl esters (FAMEs) according to slight modifications tothe method used by [30] Approximately 02 g of extractedlipid samples was diluted with 4 mL of hexane followed byaddition of 02 mL of sodium methoxide in sealed tubesThe mixture was vortex-mixed and left to stand for about 30minutes to form two layers subsequent to which the top layercontaining FAMEs was collected for fatty acids (FA) analysisGas chromatography (GC) (model COND BPX70M) fittedwith a capillary column (BPX7060 m times 025 mm) andequipped with flame ionization detector (FID) was usedApproximately 1 120583L of each H leucospilota organ extract wasinjected and separated on the GC column Splitless injectionwas also performed at 10-minute purge time Helium thatrepresented the carrier gas was calibrated at a flow rate of 16mLmin The program for column temperature used was setat 8∘Cmin to 180∘C for 10minutes and subsequently 8∘Cminto 240∘C for 10 minutes Inlet temperature was held at 250∘Cand the detector temperature was maintained at 260∘Cwhereas the oven temperature was set to 115∘C Individualcompounds in the sample were identified using mass spectraldata by comparing retention time with that obtained forauthentic laboratory standards The peak areas generatedwere quantified expressed and recorded as percentage oftotal FA detected

3 Results

31 Qualitative Profile of Bioactives in H leucospilota andExtraction Efficiency of Solvents The qualitative profile of

bioactive components extracted from sea cucumber H leu-cospilota organ samples is presented inTable 1 From the tablethe presence of saponins steroids terpenoids glycosides car-diac glycosides tannins phlobatannins phenols flavonoidsand carbohydrates in different solvent extracts of the sampledsea cucumber organs was confirmed positive although indifferent concentrations

Among the groups of compounds tested assessment forphenols terpenes and carbohydrates (polysaccharides) waspositive in all solvent extracts of all three organs evaluatedusing both conventional and Soxhlet extraction procedure(and well distributed in the organic and aqua solvents)On the other hand test for phlobatannins was negative inall solvent extracts of Cuvierian tubules (CT) and visceralorgans (VO) and all other solvent extracts used on body wall(BW) except in acetone (A) and methanolacetone (MA)indicating that the presence of phlobatannins was restrictedto the BW Screening for tannins was positive in all solventextracts of both VO and BW but was negative in extractsof CT Tests for steroids saponins cardiac glycosides andglycosides yielded positive results in solvent extracts of alltissues evaluated except those extracted in A In the case offlavonoids screening yielded positive results in all solventextracts of BW and MA extract of VO Test for flavonoidshowever yielded negative result in A extract of VO and bothsolvent extracts of CT

Results for the quality screening also indicated that theMA solvent combination was by far the most efficient extrac-tion solvent yielding all the ten groups of compounds acrossalmost all organ samples studied Conversely Awas observedas the lowest efficient extraction solvent across the organsamples studied as it was only efficient in extracting 3 groupsof compounds in CT (phenols terpenes and carbohydrates)4 groups from VO (phenols terpenes carbohydrates andtannins) and 6 groups from BW (phenols terpenes car-bohydrates tannins phlobatannins and flavonoids) Com-parison between the conventional and Soxhlet extractionprocedure from the quality assessment test was in favorof the conventional one (marked in the Table 1 by ) as


Table 2 Concentrations of groups of bioactive components in body wall (BW) extracts of sea cucumber H leucospilota sampled fromPeninsular Malaysia from a study by [19]

Compound groupConcentration of Sample Approximate

(mgg) Quantity( total)

Phenolics 458 plusmn 0002 mg GAEg 7979Flavonoids 084 plusmn 0000 mg REg 1463Saponins 032 plusmn 0002 mg DEg 558a-Values represent mean of 3 replicate determinations plusmn SDGAE gallic acid equivalent (mg GAEg) RE rutin equivalent (mg REg) DE diosgenin equivalent (mg DEg)

Phenolics (GAE) Flavonoids ( RE) Saponins (DE)Compound groups (Concentration Equivalence)

005

115

225

335

445

5

Conc

entr

atio

n (m

gg)

Figure 1 Graphical presentation of estimated concentrations ofgroups of bioactive components in body wall (BW) extracts of seacucumberH leucospilota sampled from Peninsular Malaysia GAEgallic acid equivalent RE rutin equivalent DE diosgenin equivalent(all in mgg) from study [19]

all the extraction solvents tested on the BW successfullyyielded all groups of compounds except phlobatannins likelydue to the combined use of distilled water that favoredthe extraction of water soluble bioactives ConsequentlyMA and methanoldistilled water (MD) seemed the mostefficient solvent combinations for extraction of almost all thebioactives

32 Concentrations of Major Groups of Bioactives in Hleucospilota Table 2 and Figure 1 represent results for totalphenolics saponins and flavonoids content in BWextracts ofsea cucumber H leucospilota (in different equivalents) Theapproximate content ( total) is also shown (assuming onlythe 3 groups of compounds constituted the entire bioactivesin the BW of the species) From the table total phenolicscontent was measured as 458 mgg expressed as gallic acidequivalent GAE calculated from the standard curve equa-tion y = 00013x r2 = 09958This represented approximately80 of the 3 groups of bioactives measured in the BWextract

On the other hand the content of total flavonoids wasmeasured as 084 mgg expressed as rutin equivalent REcalculated from the standard curve equation y = 00009x r2= 0996 representing approximately 15 of the bioactives inBW extract of the species Quantification of total saponinscontent yielded 032 mgg expressed as diosgenin equiva-lent DE also calculated from the standard curve equation

y = 0004x r2 = 09954 in the BW extract of H leucospilotawith the estimation representing lt6 of total of the 3 groupsof bioactives evaluated

33 Identification and Estimation ( of Compounds Detected)of Bioactives in H leucospilota The twelve major subgroupsof bioactive components detected in H leucospilota via GC-MS analysis are presented in Table 3 From the table twoketone compounds were detected from which 4-hydroxy-4-methyl-2-pentanone was the major compound detectedin all CT VO and BW solvent fractions evaluated withthe concentration being the highest in the distilled waterfraction of BW The second ketone 4-methyl-3-Penten-2-one was also detected but was limited to only solventextracts of the CT and VO and was poorly detected in BWaqueous fractions One phenolic compound was detected 24-bis (1 1-dimethylethyl)-Phenol- which was found in mostsolvent fractions of all organs tested One of the five alcoholsdetected 2-Furanmethanol was found in most fractions ofall organs Another one (glycerin) was more abundant inaqueous fractions of BW and both organic solvent extractsof VO but not in CT The highest concentration of alcoholiccompound (trans-9-Hexadec-en-1-ol 915) was detectedin the MA extract of CT The remaining two alcohols((+)-transtrans-5-caranol and 10-Methyl-8-tetradecen-1-ol-acetate) were detected in high concentrations in CT acetonefraction

Seven different hydrocarbonswere isolated andmeasuredin theH leucospilota solvent fractions two prominent amongwhich were well detected in all solvents 3-Chlorooctane(which was more in acetone extracts) and 2-Chlorooctane(distributed in A MA and aqueous extracts) Two others3 4-dimethyl-1-decene and 1-Nonene were only detectablein solvent fractions of CT and VO and poorly detectedin BW The remaining hydrocarbons 3-ethyloctane andCyclopentane-1-ethyl-3-methyl were only detectable in MAfraction of CT For FA a total of nine were detected by theGC-MS analysis Of these only two (n-hexadecanoic acidand Cyclohexane butanoic acid) were detected in solventfractions of CT whereas all the other seven were found inthe solvent extracts of both VO and BW (distributed inboth organic and aqueous fractions) The highest detectedFAwas n-hexadecanoic acid that was extracted in appreciableconcentrations in all solvent fractions of CTVO andBWThevast majority of FA detected in abundance were found in BWaqueous fractions


Table3Bioactivec

ompo

nentsd

etectedandmeasuredviaG

C-MSindifferent

solventextractso

fbod

yorgans

ofseac

ucum

berHleucospilota

sampled

from

Peninsular

Malaysia

RTCom

poun

dsof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

EDBW

MDBW

PDBW

Ketones

5558

4-hydroxy-4-methyl-2

-Pentano

ne

3715

18869

27189

25

177

308

341

5533

4-methyl-3

-Penten-2-on

e05

382

18117

19-

--

-12

2Ph

enols

20225

24-bis(11-dim

ethylethyl)-Ph

enol-

27

111

213

359

214

-095

141

173

Alcohols

13367

2-Fu

ranm

ethano

l37

105

47

053

15042

-018

067

072

20783

(+)-transtrans-5-caranol

38

--

--

--

--

-21017

10-M

ethyl-8

-tetradecen-1-ol-acetate

66

--

--

--

--

-226

trans-9-Hexadec-en-1-o

l-

915

--

--

--

-018

2035

Glycerin

--

073

05

--

097

049

654

-Hydrocarbons

564

23-Ch

lorooctane

58

337

20413

20475

-12

-367

5808

2-Ch

lorooctane

68

105

95226

1145

-366

728

836

8933

34-dimethyl-1-decene

25

402

39

-1

114

--

--

9075

3-ethyloctane

-545

--

--

--

--

5592

1-methyl-3-ethylC

yclopentane

-117

--

--

--

--

5592

Cyclop

entane1-ethyl-2-m

ethyl

--

--

519

--

--

-76

921-N

onene

11117

-059

08

--

--

-Fatty

acids

23117

Tetradecanoica

cid

--

2021

12-

116

104

093

065

25783

n-Hexadecanoica

cid

08

052

88

112

613

979

3127

568

343

26217

Z-11-Hexadecanoica

cid

--

57

-41

--

--

-26267

9-Hexadecenoica

cid

--

35

-27

079

48

324

241

167

29683

cis-9-O

ctadecenoica

cid(oleicacid)

--

045

077

05

-294

152

--

2453

3Eicosano

icacid

(arachidicacid)

--

017

051

--

132

068

022

-2713

3Heptadecano

icacid

--

--

05

--

172

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acidscontin

ued

10642

Cycloh

exaneb

utanoica

cid

-10

6-

--

--

--

-289

Octadecanoica

cid

--

1602

19057

354

372

133

085

Carboxylicacid

1065

Aceticacid

08

--

-08

036

-082

11097

14717

Croton

icacid

(2-butenoica

cid)

04

022

052

067

046

--

-016

04

Aldehydes

15267

24-

dimethylb

enzaldehyde

--

13083

26

085

-044

091

-14225

37-dimethyl-2

6-O

ctadienal

--

--

-464

--

--

1035

Furfu

ral

--

--

18083

--

--

22033

(Z)-9-

Octadecenal

--

-111

13-

--

--

Sterols

409

Stigmast-5

-en-3-ol(3120573

24120590

)-(sito

sterol)

--

05

-254

--

-215

47017

14-M

ethyl-e

rgost-8

-en-ol(3120573

5120572)-

--

--

--

077

734

--

38558

Stigmasta-522-dien-3-olacetate(312057322120577)-

--

-085

--

-17

2319

Ergosta

-1422-dien-3-olacetate(3120573

512057222120576)-

--

--

--

--

103

-309

Cholest-3-ene(5120573)

-10

8-

-36

316

--

337

356

33858

Cholesta-35-diene

-095

-043

08

263

--

229

196

37717

Ergosta

-422-diene

--

--

-19

8-

--

-35592

Ergosta

-4622-triene

--

--

04

--

--

121

Fatty

acidmethylethylesters

19592

9-Hexadecenoica

cidmethyleste

r-

336

032

128

13361

--

--

19308

Hexadecanoica

cidmethyleste

r-

786

-1763

-90

5024

--

-21225

Octadecanoica

cidmethyleste

r06

741

044

835

11482

--

025

033

21483

13-O

ctadecenoica

cidmethyleste

r-

196

-38

-17

2-

--

-2315

7-Hexadecenoica

cidmethyleste

r-

391

-212

-212

-013

-017

17783

Pentadecanoica

cidmethyleste

r-

033

-407

-17

2-

--

-19575

Hexadecenoica

cidmethyleste

r-

--

--

635

--

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acid

methylethylesterscontinued

22292

Eicosano

icacid

methyleste

r-

17-

--

107

--

--

34875

11-Eicoseno

icacid

methyleste

r-

044

02

075

-014

217

121

607

359

17233

Methyltetradecano

ate

09

087

-337

-12

1-

--

-39717

581114-Eicosatetraenoica

cidethyleste

r(arachido

nica

cid)

-827

-105

11127

206

119

604

302

40725

581114

17-E

icosapentaenoica

cidmethyleste

r(EP

A)

-255

-557

-368

--

--

42842

47101316

1719

-Docosahexaeno

icacidm

ethyleste

r(DHA)

--

--

--

274

--

-Esters

31783

Decanedioicacidbis(2-ethylhexyl)ester

4-

-28

--

--

-114

24883

Hexanedioicacidbis(2-ethylhexyl)ester

--

38

--

--

--

-17075

n-tridecylester2-Prop

enoica

cid

-91

--

--

--

--

Amines

16017

2-methyl-2

-Propanamine

--

31

085

--

--

--

16308

244-Trim

ethyl-2

-Pentanamine

--

--

28

--

--

--representsn

otdetectableA

CTaceton

eCuvieria

ntubu

leM

ACTmethano

lacetone

Cuvieriantubu

leA

VOacetone

visceralorganMAV

Om

ethano

lacetone

visceralorganABWacetone

body

wallM

ABW

methano

lacetone

body

tissuePA

BWisoprop

anolacetone

body

tissueED

BWethanoldistilled

waterbo

dywallMDBW

methano

ldistilled

waterbo

dywallPD

BWisoprop

anoldistilled

waterbo

dywallextracts


0

FAME

TPEALD

PNLCHO

ALCCBA ST FA AM

HCN KE

Compound group

5

10

15

20

25

30

35

Perc

enta

ge (

) in

extr

act

Figure 2 Concentrations () of compound subgroups detected via GC-MS and measured in BW extract of sea cucumber H leucospilotaFAME fatty acid methyl esters TPE terpenes ALD aldehydes PNL phenols CHO carbohydrates ALC alcohols CBA carboxylic acidsST sterols FA fatty acids AM amines HCN hydrocarbons KE ketones

Only two carboxylic acids (Acetic and Crotonic acids)were detectable in the GC-MS analysis Whereas the first wasonly noted in BW fractions (more in aqua fractions) thesecond was measured in both fractions of CT and VO Onfurther analysis four aldehydes were detected in total whichwere mostly not found in the MA and A fractions of CTor the isopropanolacetone (PA) isopropanoldistilled water(PD) andmethanoldistilled water (MD) fractions of BWbutwere more abundant in the A and MA fractions of VO aswell as MA fraction of BW Furfural another aldehyde wasonly found in A and MA fractions of BW and 37-dimethyl-26-octadienal was detected (464) as the most abundantbut only in MA fraction of BW The most abundantlydetected sterol compound (Cholest-3-ene (5120573)) followed byCholesta-35-diene was abundant in solvent fractions (exceptA) of the 3 organs Four other sterols detected were foundin different solvent fractions of different organs where 14-methyl-ergost-8-en-ol (3120573 5120572) was the sterol detected inthe largest quantity (774) in the PD (aqueous) fraction ofBW

FAMEs and some fatty acid ethyl esters were the groupof compounds that was extracted in the highest concen-tration being found abundant in especially MA fractionsof CT VO and BW but poorly detected in A fractionThe FAMEs that were detected in the highest concentrationwere five hexadecanoic acid methyl ester octadecanoic acidmethyl ester 7-hexadecenoic acid methyl ester 581114-eicosatetraenoic acid ethyl ester (arachidonic acid) and58111417-eicosapentaenoic acid methyl ester (EPA) Con-versely 471013161719-docosahexaenoic acid methyl ester(DHA) was only found in PA fraction of BW Interestingly11-eicosenoic acid methyl ester arachidonic acid and DHAwere more abundant in aqueous fractions of BWThree estercompounds were detected in the sea cucumber extractsfound in CT and VO fractions and only one found in PDfraction of BW (Decanedioic acid bis (2-ethylhexyl)ester)as the most abundant ester detected was n-tridecyl ester2-Propenoic acid (91) in MA fraction of VO Finally

only 2 amines were extracted and detected (2-methyl-2-Propanamine and 244-Trimethyl-2-Pentanamine) both ofwhich were not detected in CT but in both fractions of VOand only one in A fraction of BW

A summarized comparison of the quantitative detec-tion levels of the twelve different subgroups of bioactivecompounds in the BW solvent extracts of sea cucumber isshown graphically in Figure 2 From the figure it would beobserved that FAMEs constituted significantly (Plt005) themost detected compounds and accounted for approximately30 of all bioactive subgroups This was followed by hydro-carbons that accounted for over 15 and then ketones andFA that accounted for lt15 each Alcohols sterols terpenesaldehydes and phenols were all detected within the rangeof 5 or less However the lowest subgroup of compoundsdetected was carboxylic acids (CBA) followed by amines(AM) and then carbohydrates (CHO) in the decreasing orderCBA gt AM gt CHO and all 3 were detected within the rangeof 1 or less of the total bioactives measured

Overall when fatty acids and FAMEs (together with fattyacid ethyl esters) are considered as one group of bioactivesthis automatically makes them account for approximately50 of all bioactives in the BW extracts of the sea cucumberspecies sampled

34 Concentration ( of Extracts) of FAMEs in BW Samplesof Sea Cucumber via GC-FID Analysis The GC-FID analysisof the FA detected in three BW solvent extracts of seacucumber H leucospilota yielded the results presentedin Table 4 From the table of the three different solventfractions tested (based on relative abundance of PUFAin these solvents in the GC-MS data) saturated FAMEsaccounted for 3707 5789 and 5318 of the FAMEs extractedin ethanoldistilled water (ED) methanoldistilled water(MD) and isopropanoldistilled water (PD) fractionsrespectively Methyl laurate (C120) methyl myristate (140)methyl palmitate (C160) and methyl stearate (180) werethe principal saturated FA with C160 being the most


Table 4 Quantitative profile of major FAMEs detected via GC-FID and measured in body wall (BW) solvent extracts of sea cucumber Hleucospilota sampled fromMalaysian peninsular

Fatty acid name No of C-atoms Tissue extract( of total extract) EDBW MDBW PDBWmethyl octanoate C80 164 plusmn 000 000 plusmn 000 000 plusmn 000methyl decanoate C100 000 plusmn 000 043 plusmn 022 000 plusmn 000methyl laurate C120 1041plusmn 167 0604 plusmn 009 000 plusmn 000methyl tridecanoate C130 000 plusmn 000 000 plusmn 000 000 plusmn 000methyl myristate C140 404 plusmn 000 0886 plusmn 000 1071 plusmn 047methyl pentadecanoate C150 064 plusmn 000 0048plusmn 0001 0808plusmn 002methyl palmitate C160 1481 plusmn 066 4486 plusmn 339 26477plusmn 062methyl heptadecanoate C170 0379 plusmn 173 0138 plusmn 001 0885 plusmn 002methyl stearate C180 483 plusmn 041 9516 plusmn 182 6835 plusmn 022methyl arachidate C200 0321 plusmn 000 1314 plusmn 014 1189 plusmn 008methyl behenate C220 000 plusmn 000 009 plusmn 004 033 plusmn 016erucic acid methyl ester C230 000 plusmn 000 000 plusmn 000 5949 plusmn 030Total Saturate d FA (total FA) 3707 5789 5318cis-9-oleic methyl ester C181 450 plusmn 708 2638 plusmn 328 2229 plusmn 047trans-9-elaidic methyl ester C191 000 plusmn 000 000 plusmn 000 000 plusmn 000methyl eicosenoate C201 020 plusmn 000 0134 plusmn 001 171 plusmn 026Total mono un saturate d FA ( total FA) 4520 2651 2400methyl linoleate C182 1479 plusmn 351 1557 plusmn 163 11107 plusmn 014methyl linolenate C183 040 plusmn 000 0368 plusmn 002 0566 plusmn 007Total PUFA (total FA) 1519 1594 1167aValues represent mean of 3 replicate determinations plusmn standard deviationEDBW ethanoldistilled water MDBW methanoldistilled water PDBW isopropanoldistilled water

abundant Monounsaturated FA on the other handaccounted for 4520 2651 and 2400 of total FAMEsextracted in ED MD and PD fractions respectively and theprincipal monounsaturated FA across the 3 solvent extractswas cis-9-oleic methyl ester (C181) Polyunsaturated (PUFA)FAMEs were also detected by the solvent extracts used andmeasured as 1519 1594 and 1167 in the solvent fractionsstudied respectively The most abundant PUFA FAME wasC182 (methyl linoleate)

Generally a comparison among the solvents used onBW extracts revealed that MD and PD were more efficientin extracting saturated FA (5789 and 5318) and ED wasmore efficient in extracting monounsaturated FA (4520)whereas all three solvents exhibited almost equivalent effi-ciency for extracting PUFA FAMEs from BW of the seacucumber (1519 1594 and 1167 respectively)

4 Discussion

41 Qualitative Analysis for Groups of Compounds In gen-eral detection of the different bioactives in the differentorgan extracts evaluated was influenced by the polarity ofsolvents and sensitivity of the extraction methods employedIt is already well established that highly polar solventslike methanol ethanol isopropanol (in lipids) and water(in aqueous medium) have higher capacity to extract highconcentrations of polar compounds (such as phenolicsflavonoids and some saponins) whereas solvents of middle

to lower polarity yield different concentrations of bioactivesof middle to lower polarity from both plant [31] and animalmatrixes [32 33] This observation correlates with phenolicsflavonoids and saponins being the most abundant bioac-tives (in that decreasing order) extracted in the methanolicethanolic and aqueous fractions of H leucospilota organextracts Many other authors have reported different concen-trations of these bioactives in different organs and tissues ofdiverse solvent extracts of sea cucumbers including phenolsand tannins [14 34] phenols and flavonoids [26] So farmost marine polyphenols referenced in the literature wereisolated and identified as having originated frommacroalgaeresources [35] over 8000 different phenolic compounds arecurrently known [36] and many are likely to be discovered

Chemical extraction and screening constitute a veryuseful method to determine the presence and concentrationsof bioactive constituents in organisms [37] Analysis of someisolated bioactive compounds inH leucospilota indicates thatthey may be completely harmful to humans and other marineorganisms as the substances may have been absorbed intothe tissues of the sea cucumber from toxic wastes regularlydumped into the sea especially heavy metals like CadmiumArsenic and Lead [38] On the other hand some otherdetected substances may help to protect the sea cucumberagainst harmful bacteria and predators abundant in themarine environment [39 40] Consistent with this specu-lation a study [41] confirmed that certain isolated organicsubstances from organs of sea cucumbers were active against


both Gram-positive (Bacillus subtilis Staphylococcus aureus)and Gram-negative bacteria (Escherichia coliKlebsiella pneu-moniae) [42] and methanolic extracts of sea cucumbershave also been shown to exhibit cytotoxicity and antifungalactivities against Aspergillus niger [43] Furthermore theshallow reef areas where H leucospilota is commonly foundare also home to some predators of the species making itnecessary for the organism to devise strategies to protectitself and may therefore justify accumulation or synthesis ofprotective poisons [44]

42 Concentration of Compound Groups in BW Extract ofH leucospilota The presence of phenolics saponins andflavonoids in H leucospilota extracts is indication of themedicinal importance of the species These groups of com-pounds are known to possess healing properties and have alsobeen demonstrated to be active against several pathogensthus supporting the traditional use of sea cucumbers for themanaging several diseases Saponins are known to exhibita wide spectrum of biological effects including hemolyticcytostatic antineoplastic anticancer and antitumor activi-ties [45ndash48] Studies reported by other authors have alsodemonstrated the antioxidant properties of sea cucumbersthat were described to possess high content of phenolics andflavonoids where total flavonoids from the gonads musclesand digestive tract were noted to constitute the highestrepository [8] Tannins a group of phenolic metabolites thatare found in many terrestrial plants [49] are also knownto be abundant in marine resources like macroalgae andsea cucumbers because of their high antioxidant capacity[50]

The largest of the compounds of plants phenolics areflavonoids that account for about 50 of the 8000 naturallyoccurring phenolic compounds [51] Many studies have beencarried out on terrestrial plants-derived flavonoids but thoseon the algal-derived flavonoids are scarce Consequently ithas been shown that there are basic structural (and likelyfunctional) differences between flavonoids frommarine algaeand those in terrestrial vegetables and fruits [52] as [53] con-firmed that macroalgae are rich sources of the catechins andflavonols the rare types of flavonoids which sea cucumbersare likely to have in abundance because they feed mainlyon seaweeds These compounds likely constitute part of theapproximate quantity ( total dryweight value) of flavonoidsrecorded in the present study (146) which was similar tothat reported elsewhere (12) [54]

Saponins commonly identified as holothurins from seacucumber with structural features quite comparable to thoseinGanoderma Ginseng and other medicinally popular tonicherbs have also been isolated by other authors [55] Thiswater soluble group of substances is usually found amongmarine species such as sea cucumbers star fish and spongeswith triterpene glycosides reported as predominant amongthese compounds in sea cucumber The chemical diversityof triterpene holothurins from sea cucumbers makes themcurrently the attractive group of compounds targeted in newdrugs discovery [56] Mucopolysaccharides and chondroitinhave also been detected in sea cucumbers and have proveduseful as nutraceuticals to people suffering from arthritis and

connective tissue disorders to ease joint-pains and arthritis[57]

Detection of phlobatannins in solvent fractions of seacucumber body walls was also not a surprise Phlobatanninswhich are polymers of phloroglucinol are known structuralcomponents of brown algal cell wall that offer protec-tion to the algae against harmful ultraviolet radiation [35]Phlorotannins unlike hydrolysable or condensed tanninsare oligomers of phloroglucinol and are restricted to brownmarine algae [58] They also taste bitter and help the algaeto avoid foragers [59] Sea cucumbers consume macroalgaeas their major source of nourishments [13] and may haveaccumulated the phlobatannins likely to serve as protectionagainst microbial infection or to also deter predators

Regardless of their potential bioavailability is the majorchallenge confronting researchers when questions about therelationships existing between polyphenols and their healthbenefits are raised [60] In other words the exact in vivoeffects of consumed polyphenols (either bymarine organismsor humans) are yet to be fully understood as research on thetopic is still ongoing with irregular and vague conclusionsbetween studies when compared to what is known in vitroHowever the potential bioavailability of polyphenolics suchas phlorotannins from marine algae indicated that over 70of the consumed plant polyphenols evaluated in vivomay bebioavailable and utilizable for their presumedmetabolic roles[61]

43 Bioactive Compounds and Their Concentrations ( ofExtract Detected) in H leucospilota Data in the literaturesuggests that the compound 2-pentanone 4-hydroxy-4-methyl- one of the compounds found in sea cucumberhas also been isolated from species of red and brown algae(Rhodophyceae and Phaeophyceae) and also in algal speciesof Laurencia pinnatifida and Pterocladia capillacea Thesesubstances were reported to be responsible for the antimi-crobial and antioxidant activities of these algae [62] Thepresence of high concentrations of these compounds in Hleucospilota is likely due to dependence of sea cucumberson phytoplankton and micro- and macroalgae as their majorsources of nourishment [13] Furthermore the detection ofhexadecanoic acid and 58114-eicosatetraenoic acid ethylester (arachidonic acid ethyl ester 204n6) as other com-pounds found in high concentration in all extracts wasnot surprising as arachidonic acid a long-chain fatty acidtogether with eicosapentaenoic acid (EPA 205n3) anddocosahexaenoic acid (DHA 226n3) has been shown tobe synthesized by some marine bacteria and phytoplanktonand transferred through the food web to other organismsincluding sea cucumbers that feed on them [63 64]

These omega 3 and omega 6 FA are essential in humannutrition and are currently highly sought after even in animalhusbandry The detection of these major components in thetissues of sea cucumber was therefore consistent with itsfeeding habits as reported by [13] additional reason for itsconsumption as food Other bioactive and antiagent sub-stances in sea cucumbers such as triterpene glycosides someenzymes amyloses fatty acids and cytotoxins that havepotential to improve immunity resist tumor offer protection


to nerve tissue reduce pain and contribute to immunepotentiation anticancer and anticoagulation activities havealso been properly documented [65]

Revelation by GC-FID analysis that H leucospilota organextracts contained FAMEs as the most abundant (approxi-mately 50) subgroup of bioactives consisting of saturatedFA (methyl palmitate methyl laurate methyl stearate anderucic acid methyl ester) monounsaturated FA (cis-9-oleicmethyl ester and methyl linoleate) and PUFA (Arachidonicacid EPA) as some of the important compounds wasexpectedThe sea cucumberH leucospilota is a bottom feederon sea sediments that is expected to contain high concentra-tions of FA This is because sediments at sea bottoms thatthe species feeds on are known to contain relatively highcontents of branched chain FA [66ndash68] The origin of thesediments is believed to be mostly from bacteria althoughhigh levels of algal-derived FA have also been reported inseveral species of sea cucumbers Micro- and macroalgae arebelieved to be the main sources of plant materials in the dietfor demersal animals in shallow (lt100 m) coastal waters thatare grazed directly by herbivorous and omnivorous species[69] Similarly micro- and macroalgae are well known asproducers of hydrocarbons consequently hydrocarbons areexpected as constituents of demersal animals that depend onthese algae as their sources of energy On the other handketones found in H leucospilota were also found in certainalgal species andmay have been transferred through the foodweb

Organic matter synthesized in the marine ecosystem byphotosynthetic organisms is utilized either directly in thefood web or through the microbial loop [70] Photosyntheticorganisms are themselves consumed in the food web andeventually supply essential FA to marine organisms whichare incapable of synthesizing them in sufficient quantities tocover their physiological needs [71] More pertinently onlyplants have demonstrated capacity for de novo synthesis of183n-3 and 182n-6 FA in marine ecosystems Consequentlymicro- and macroalgae primarily produce omega-3 andomega-6 polyunsaturated fatty acids (PUFA) which areeventually transferred via the food chain to higher animalsthat feed upon them [72 73]

Reference [74] opined that the percentage of contentof saturated and unsaturated fatty acid esters in animalscould also be used as indication of the feeding habits oforganisms The dominance of saturated FA would indicateautotrophic feeding whereas abundance of unsaturated FAindicated heterotrophic feeding implying that the organismrelied more on zooplanktons and small fishes The laterdefinition is consistent with the FA profile detected in Hleucospilota Monounsaturated fatty acids (MUFA) such asoleic acid and polyunsaturated fatty acids (PUFA) such asarachidonic acid (AA) eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) are essential FA required forgood human health and nutrition These FA are required forprotection from the risk of coronary heart diseasesThey playimportant roles in the lives of cardiac cells as they are essentialfuels for the mechanical and electrical activities of the heart[75] SFA such as hexadecanoic acid octadecanoic acid andtetradecanoic acid were also found in the samples evaluated

although in low concentrations These FA are known toraise LDL cholesterol and thus increase the risk of coronaryheart disease However octadecanoic acid (stearic acid) hasbeen shown to have a neutral effect on total blood and LDLcholesterol levels [76ndash80] It is not coincidence therefore thatthe heavy consumption of sea cucumbers over the centurieswas for nothing other than the health and nutritional benefitsrealized by ancient generations who passed down the habit totheir descendants

Other substances found in the solvent extracts have beendemonstrated to have biological activity For instance triter-pene (a terpenoid) compound was proven to exhibit cyto-toxicity against some cancer cells including human tumorcell lines as a potential anticancer agent Another com-pound intercedenside A also demonstrated antineoplasticfunction against mouse sarcoma and mouse lung cancer [81]A study in Japan stumbled upon some novel compounds(gangliosides) in a sea cucumber species H leucospilotawhich were able to stimulate laboratory-scale nerve cellgrowth in rat cells [82] The potential to use compounds likechondroitin sulfate from sea cucumbers for the inhibitionof human immunodeficiency virus (HIV) infection has alsobeen proposed as sulfated polysaccharides such as fucoidanisolated from seaweeds and sea cucumbers were reported toexhibit antibacterial anticancer and antiviral activities [83]Polypeptides and polypeptides in sea cucumber extracts havealso been shown to exhibit free radical scavenging abilityand antioxidant abilities according to studies reported by[84] and [85] respectivelyThenumber of potential biologicalactivities of compounds extractable from sea cucumbers isstill evolving

44 FAMEs ( of Extracts Detected) in BW Samples of SeaCucumber Quantified Reference [86] concluded that fattyacids of sea cucumber lipids fractions were the key compo-nents liable for tissue repair and wound healing properties ofsea cucumber Palmitic acid has been reported to constitutebetween 20 and 30 of most animal fats and also animportant constituent of most vegetable fats (35ndash45 ofpalm oil) Stearic acid (octadecanoic acid) is naturersquos mostcommon long-chain FA found in combined form in naturalanimal and vegetable fats [87] In contrast to vegetable oilshowever FA with odd number of carbon (C130 170 C190)are also found in sea cucumbers as was also evident from theresults of the present study [38] Among the unsaturated fattyacids cis-9-oleic methyl ester (C181n9) a MUFA and methyllinoleate (C182n6) an omega-6 PUFA also formed themajorconstituents of the lipids Cis-9-oleic methyl ester (cis-9-octadecenoic acid) detected as the highest concentration inone of the extracts is reported to be themost abundantMUFAin animal tissues Methyl linolenate (C183n3) an omega-3PUFA is commonly found in low concentrations in manyanimal tissues [15]

Deposit feeding holothurians (among which are seacucumbers) are reported to possess greater amounts of algal-derived FA such as arachidonic acid DHA and EPA Theprincipal FA of holothurians comprised C204 (n-6) C201C205 (n-3) C160 and C180 and also high content of otherFA such as C181 C200 C231 and C226 (n-3) The results


of the present study further showed that more saturated fattyacids were found in H leucospilota compared to the unsat-urated FA Among the PUFA in sea cucumbers arachidonicacid (C204 n-6) is detected to be the principal componentin almost all species with relatively higher amounts reportedfor tropical species as was observed in the present study [88]This FA is known to play an important role in the processesof cell growth and clotting of blood leading to healing ofwounds [79] and lending support to the long-time utilizationof sea cucumbers in Asia as traditional remedy to treat burnsand cuts [89]

High level of arachidonic acid (AA) was detected inH leucospilota via GC-MS analysis AA is known to beresponsible for blood clotting [90] and was the main PUFAdetected in all the samples evaluated In tropical holothuriansthe level of AA usually prevails appreciably over that forEPA [16] Oleic acid the major MUFA on the other handis associated with the prevention of coronary heart diseaseand was found to be the highest in GC-FID analysis Themajor omega-3 FA found in H leucospilota point to itsimportance and use in traditional medication for cure and inthe prevention of many illnesses and infections The resultstherefore indicate that H leucospilota is particularly rich inMUFA PUFA and amino acids that compete with levelsfound in some of the commercially important species of seacucumber in terms of nutritional and medicinal values

5 Conclusion

In conclusion from the results of the investigations carriedout in this study it is clear that H leucospilota is a potentialsource of many bioactive nutritionally and physiologicallyimportant compounds that could be utilized in nutraceuticaland pharmaceutical industries as sources of both food andmedicine However further studies need to be carried out toisolate purify and specifically investigate the efficacies andbiological activities of all the identified compounds and thecurrent focus on the species tomine for new drugs is justified

Data Availability

The data and materials supporting the conclusions of thisarticle are included within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The first author expresses deep appreciation to the IslamicDevelopment Bank Group (IDB) for the financial support hereceived in the form of scholarship to enable the completionof his MS studies All authors express deep appreciation tothe Institute of Bioscience (IBS) Universiti Putra Malaysia(UPM) for providing the facilities which enabled the com-pletion of this research work funded under FundamentalResearchGrants (02-04-10-881 FR) and theHigher EducationCentre of Excellence (HiCOE) (6369100) by the Malaysian

Ministry of Higher Education (MOHE) the University PutraMalaysia Research University Grants (05-05-10-1075RU)

References

[1] A M Kerr and J Kim ldquoPhylogeny of holothuroidea (echino-dermata) inferred from morphologyrdquo Zoological Journal of theLinnean Society vol 133 no 1 pp 63ndash81 2001

[2] C Conand and M Byrne ldquoA review of recent developments inthe world sea cucumber fisheriesrdquoMarine Fisheries Review vol55 no 4 pp 1ndash13 1993

[3] A W Bruckner K A Johnson and J D Field ldquoConservationstrategies for sea cucumbers Can a CITES Appendix II listingpromote sustainable international traderdquo SPC Beche-de-merInformation Bulletin vol 18 pp 24ndash33 2003

[4] S Jaquemet and C Conand ldquoThe beche-de-mer trade in19951996 and an assessment of exchanges between main worldmarketsrdquo SPC Beche-de-mer Information Bulletin vol 12 pp 11ndash14 1999

[5] H Benedikt ldquoThe therapeutic benefits of sea cucumber (bechede mer)rdquo Chiropractic Products pp 104ndash107 1997

[6] Food and Agriculture Organization of the United NationsldquoBrief introduction to mariculture of five selected speciesin Chinardquo UNDPFAO Regional Seafarming Developmentand Demonstration Project (RAS90002) National InlandFisheries Institute Kasetsart University Campus BangkhenBangkok Thailand 1990

[7] R P Vieira and P A Mourao ldquoOccurrence of a unique fucose-branched chondroitin sulfate in the body wall of a sea cucum-berrdquo The Journal of Biological Chemistry vol 263 no 34 pp18176ndash18183 1988

[8] S Bordbar F Anwar and N Saari ldquoHigh-value componentsand bioactives from sea cucumbers for functional foodsmdashareviewrdquoMarine Drugs vol 9 no 10 pp 1761ndash1805 2011

[9] J A Findlay and A Daljeet ldquoFrondogenin a new aglyconefrom the sea cucumber cucumaria frondosardquo Journal of NaturalProducts vol 47 no 2 pp 320ndash324 1984

[10] C Conand FAO Species Identification GuideThe Marine LivingResources of the Western Central Pacific Vol 2 CephalopodsCrustaceans Holothurians And Sharks K Carpenter and VNiem Eds vol 2 Cephalopods Crustaceans Holothuriansand Sharks 1998

[11] Y Samyn D Van den Spiegel and C Massin Taxonomie desholothuries des Comores ABC Taxa Volume 1 Indash III RoyalBelgian Institute of Natural Sciences Brussels Belgium 2006

[12] C Conand ldquoPopulation status fisheries and trade of seacucumbers in Africa and Indian oceanrdquo in Sea cucumbers Aglobal review on fishery and trade FAOFisheries Technical PaperNo 516 Toral-Granda V A Lovatelli and M VasconcellosEds pp 153ndash205 FAO Rome Italy 2008

[13] G R Allen and R Steene Indo-Pacific Coral Reef Field GuideTropical Reef Research Singapore 1994

[14] O Y Althunibat R B Hashim M Taher J M Daud M-AIkeda and B I Zali ldquoIn vitro antioxidant and antiproliferativeactivities of three Malaysian sea cucumber speciesrdquo EuropeanJournal of Scientific Research vol 37 no 3 pp 376ndash387 2009

[15] M Yahyavi M Afkhami A Javadi et al ldquoFatty acid com-position in two sea cucumber species Holothuria scabra andHolothuria leucospilota from Qeshm Island (Persian Gulf)rdquoAfrican Journal of Biotechnology vol 11 no 12 pp 2862ndash28682012


[16] V I Svetashev V S Levin C N Lam and D T Nga ldquoLipidand fatty acid composition of holothurians from tropical andtemperate watersrdquoComparative Biochemistry and Physiology mdashPart B Biochemistry and vol 98 no 4 pp 489ndash494 1991

[17] K Yamada R Matsubara M Kaneko T Miyamoto and RHiguchi ldquoConstituents of holothuroidea 10 Isolation andstructure of a biologically active ganglioside molecular speciesfrom the sea cucumber Holothuria leucospilotardquo Chemical ampPharmaceutical Bulletin vol 49 no 4 pp 447ndash452 2001

[18] T Matsuno and M Tsushima ldquoComparative biochemical stud-ies of carotenoids in sea cucumbersrdquoComparative Biochemistryand Physiology mdash Part B Biochemistry and vol 111 no 4 pp597ndash605 1995

[19] A Ceesay Constituents of sea cucumber Holothuria (Merten-siothuria) leucospilota Brandt with broad antibacterial activities[Masters thesis] Universiti Putra Malaysia 2013

[20] N VThanh N H Dang P V Kiem N X Cuong H T Huongand C V Minh ldquoA new triterpene glycoside from the seacucumber holothuria scabra collected in Vietnamrdquo ASEANJournal on Science and Technology for Development vol 23 no4 pp 253ndash260 2008

[21] O Oloyede ldquoChemical profile of unripe pulp of carica papayardquoPakistan Journal of Nutrition vol 4 no 6 pp 379ndash381 2005

[22] A Sofowara Medical Plants and Tropical Medicine in AfricaSpectrum Books LTD Ibadan Nigeria 1993

[23] G Trease andW Evans Pharmacognosy A Physicians Guide toHerbal Medicine Bailliere Tindall London UK 1989

[24] J B Harborne Phytochemical Methods Chapman and HallLondon UK 1st edition 1973

[25] D Krishnaiah T Devi A Bono and R Sarbatly ldquoStudies onphytochemical constituents of six Malaysian medicinal plantsrdquoJournal of Medicinal Plants Research vol 3 no 2 pp 67ndash722009

[26] J Mamelona E Pelletier K Girard-Lalancette J Legault SKarboune and S Kermasha ldquoQuantification of phenolic con-tents and antioxidant capacity of Atlantic sea cucumberCucumaria frondosardquo Food Chemistry vol 104 no 3 pp 1040ndash1047 2007

[27] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[28] H P S Makkar and K Becker ldquoPotential of J curcas mealas a protein supplement to livestock feed constraints to itsutilization and possible strategies to overcome constraintsrdquo inBiofuels and Industrial Products from Jatropha curcas G MGubitz M Mittelbach and M Trabi Eds pp 190ndash205 DBVGraz 1997

[29] H Han Y-H Yi L Li et al ldquoTriterpene glycosides from seacucumber Holothuria leucospilotardquo Chinese Journal of NaturalMedicines vol 7 no 5 pp 346ndash350 2009

[30] R E Timms ldquoArtifact peaks in the preparation and gas-liquidchromatographic determination of methyl estersrdquo AustralianJournal of Dairy Technology vol 33 no 1 pp 4ndash6 1978

[31] M Bimakr R A Rahman F S Taip et al ldquoComparison ofdifferent extraction methods for the extraction of major bioac-tive flavonoid compounds from spearmint (Mentha spicata L)leavesrdquo Food andBioproducts Processing vol 89 no 1 pp 67ndash722011

[32] M Y Heng S N Tan J W H Yong and E S Ong ldquoEmerginggreen technologies for the chemical standardization of botan-icals and herbal preparationsrdquo TrAC - Trends in AnalyticalChemistry vol 50 pp 1ndash10 2013

[33] P C Veggi J Martinez and M A Meireles ldquoFundamentalsof microwave extractionrdquo in Microwave-Assisted Extraction forBioactive Compounds Theory and Practice F Chemat and GCravotto Eds Food Engineering Series 4 pp 15ndash52 SpringerNew York NY USA 2013

[34] R G Pacheco C P Vicente P Zancan and P A S MouraoldquoDifferent antithromboticmechanisms among glycosaminogly-cans revealed with a new fucosylated chondroitin sulfate froman echinodermrdquo Blood Coagulation amp Fibrinolysis vol 11 no 6pp 563ndash573 2000

[35] Y X Li I Wijesekara Y Li and S K Kim ldquoPhlorotannins asbioactive agents from brown algaerdquo Process Biochemistry vol46 no 12 pp 2219ndash2224 2011

[36] L Bravo ldquoPolyphenols chemistry dietary sources metabolismand nutritional significancerdquo Nutrition Reviews vol 56 no 11pp 317ndash333 1998

[37] S Sasidharan Y Chen D Saravanan K M Sundram andL Yoga Latha ldquoExtraction isolation and characterization ofbioactive compounds from plantsrsquo extractsrdquo African Journal ofTraditional Complementary and Alternative Medicines vol 8no 1 pp 1ndash10 2011

[38] J Wen and C Hu ldquoElemental composition of commercial seacucumbers (holothurians)rdquo Food Additives amp ContaminantsPart B Surveillance vol 3 no 4 pp 246ndash252 2010

[39] T Rob Aquarium Invertebrates Sea Cucumbers - Part II 2003httpwwwadvancedaquaristcom20031inverts

[40] J R Pawlik ldquoMarine invertebrate chemical defensesrdquo ChemicalReviews vol 93 no 5 pp 1911ndash1922 1993

[41] B T S Yff K L Lindsey M B Taylor D G Erasmus and AK Jager ldquoThe pharmacological screening of Pentanisia prunel-loides and the isolation of the antibacterial compound palmiticacidrdquo Journal of Ethnopharmacology vol 79 no 1 pp 101ndash1072002

[42] B Farjami M Nematollahi Y Moradi and M NazemildquoDerivation of extracts from Persian Gulf sea cucumber(Holothuria leucospilota) and assessment of its antifungaleffectrdquo Iranian Journal of Fisheries Sciences vol 13 no 4 pp785ndash795 2014

[43] F Mohammadizadeh M Ehsanpor M Afkhami A MokhlesiA Khazaali and S Montazeri ldquoEvaluation of antibacterialantifungal and cytotoxic effects of Holothuria scabra from theNorthCoast of the PersianGulfrdquo Journal deMycologie Medicalevol 23 no 4 pp 225ndash229 2013

[44] R Piper Extraordinary Animals An Encyclopedia of CuriousandUnusual Animals Greenwood PressWestport Conn USA2007

[45] G Caulier S Van Dyck P Gerbaux I Eeckhaut and P Flam-mang ldquoReview of saponin diversity in sea cucumber belongingto the family Holothuriidaerdquo SPC Beche-de-mer InformationBulletin vol 31 pp 48ndash54 2011

[46] S Van Dyck P Gerbaux and P Flammang ldquoQualitative andquantitative saponin contents in five sea cucumbers from theIndian oceanrdquoMarine Drugs vol 8 no 1 pp 173ndash189 2010

[47] F Tian C-H Zhu X-W Zhang et al ldquoPhilinopside E a newsulfated saponin from sea cucumber blocks the interactionbetween kinase insert domain-containing receptor (KDR) and120572v1205733 integrin via binding to the extracellular domain of KDRrdquoMolecular Pharmacology vol 72 no 3 pp 545ndash552 2007


[48] F Tian X Zhang Y Tong et al ldquoPE a new sulfated saponinfrom sea cucumber exhibits anti-angiogenic and anti-tumoractivities in vitro and in vivordquo Cancer Biology amp Therapy vol4 no 8 pp 874ndash882 2005

[49] T Okuda and H Ito ldquoTannins of constant structure in medic-inal and food plants-hydrolyzable tannins and polyphenolsrelated to tanninsrdquoMolecules vol 16 no 3 pp 2191ndash2217 2011

[50] Y Zhong M A Khan and F Shahidi ldquoCompositional charac-teristics and antioxidant properties of fresh and processed seacucumber (Cucumaria frondosa)rdquo Journal of Agricultural andFood Chemistry vol 55 no 4 pp 1188ndash1192 2007

[51] J B Harborne and C A Williams ldquoAdvances in flavonoidresearch since 1992rdquo Phytochemistry vol 55 no 6 pp 481ndash5042000

[52] Y Yoshie-Stark Y P Hsieh and T Suzuki ldquoDistribution offlavonoids and related compounds from seaweeds in JapanrdquoJournal of Tokyo University of Fisheries vol 89 pp 1ndash6 2003

[53] S-J Heo E-J Park K-W Lee and Y-J Jeon ldquoAntioxidantactivities of enzymatic extracts from brown seaweedsrdquo Biore-source Technology vol 96 no 14 pp 1613ndash1623 2005

[54] K T Lim C Hu and D D Kitts ldquoAntioxidant activity of aRhus verniciflua Stokes ethanol extractrdquo Food and ChemicalToxicology vol 39 no 3 pp 229ndash237 2001

[55] J Chen ldquoOverview of sea cucumber farming and sea ranchingpractices in Chinardquo SPC beche-de-mer Information Bulletin vol18 pp 18ndash23 2003

[56] Y Bahrami W Zhang and C M M Franco ldquoDistribution ofsaponins in the sea cucumber holothuria lessoni the body wallversus the viscera and their biological activitiesrdquoMarine Drugsvol 16 no 11 pp 1ndash30 2018

[57] T Imanari Y Washio Y Huang H Toyoda A Suzukiand T Toida ldquoOral absorption and clearance of partiallydepolymerized fucosyl chondroitin sulfate from sea cucumberrdquoThrombosis Research vol 93 no 3 pp 129ndash135 1999

[58] M A Ragan and K W Glombitza ldquoPhlorotannins brown algalpolyphenolsrdquo in Progress in Phycological Research F E Roundand D J Chapman Eds Biopress Ltd Bristol UK 1986

[59] H Pavia G Cervin A Lindgren and P Aberg ldquoEffects of UV-B radiation and simulated herbivory on phlorotannins in thebrown alga Ascophyllum nodosumrdquo Marine Ecology ProgressSeries vol 157 pp 139ndash146 1997

[60] MDrsquoArchivio C Santangelo B Scazzocchio et al ldquoModulatoryeffects of polyphenols on apoptosis induction relevance forcancer preventionrdquo International Journal of Molecular Sciencesvol 9 no 3 pp 213ndash228 2008

[61] G Williamson and C Manach ldquoBioavailability and bioefficacyof polyphenols in humans II Review of 93 interventionstudiesrdquo American Journal of Clinical Nutrition vol 81 no 1pp 243Sndash255S 2005

[62] S Wefky and M Ghobrial ldquoStudies on the bioactivity ofdifferent solvents extracts of selectedmarinemacroalgae againstfish pathogensrdquo Research Journal of Microbiology vol 3 no 12pp 673ndash682 2008

[63] M R Brown G A Dunstan S W Jeffrey J K Volkman SM Barrett and J LeRoi ldquoThe influence of irradiance on thebiochemical composition of the Pyrmnesiophyte Isochrysis sp(clone t-iso)rdquo Journal of Phycology vol 29 no 5 pp 601ndash6121993

[64] J K Volkman S W Jeffrey P D Nichols G I Rogers andC D Garland ldquoFatty acid and lipid composition of 10 speciesof microalgae used in mariculturerdquo Journal of ExperimentalMarine Biology and Ecology vol 128 no 3 pp 219ndash240 1989

[65] X Yuan H Yang L Wang Y Zhou and H R Gabr ldquoEffectsof salinity on energy budget in pond-cultured sea cucumberApostichopus japonicus (Selenka) (Echinodermata Holothu-roidea)rdquo Aquaculture vol 306 no 1-4 pp 348ndash351 2010

[66] R F Leo and P L Parker ldquoBranched-chain fatty acids insedimentsrdquo Science vol 152 no 3722 pp 649-650 1966

[67] J R Sargent C C E Hopkins J V Seiring and A YoungsonldquoPartial characterization of organic material in surface sedi-ments from Balsfjorden northern Norway in relation to itsorigin and nutritional value for sediment-ingesting animalsrdquoMarine Biology vol 76 no 1 pp 87ndash94 1983

[68] N W Phillips ldquoRole of different microbes and substrates aspotential suppliers of specific essential nutrients to marinedetritivoresrdquo Bulletin of Marine Science vol 35 no 3 pp 283ndash298 1984

[69] G A Dunstan A J Sinclair K OrsquoDea and J M NaughtonldquoThe lipid content and fatty acid composition of variousmarinespecies from southern Australian coastal watersrdquo ComparativeBiochemistry and Physiology mdash Part B vol 91 no 1 pp 165ndash1691988

[70] F Azam T Fenchel J Field J Gray L Meyer-Reil and FThingstad ldquoThe ecological role of water-column microbes inthe seardquo Marine Ecology Progress Series vol 10 pp 257ndash2631983

[71] T Watanabe C Kitajima and S Fujita ldquoNutritional values oflive organisms used in Japan for mass propagation of fish AreviewrdquoAquaculture vol 34 no 1-2 pp 115ndash143 1983

[72] A J Fraser J R Sargent J C Gamble and D D SeatonldquoFormation and transfer of fatty acids in an enclosed marinefood chain comprising phytoplankton zooplankton and her-ring (Clupea harengus L) larvaerdquoMarine Chemistry vol 27 no1-2 pp 1ndash18 1989

[73] J R Sargent and K J Whittle ldquoLipids and hydrocarbons in themarine food webrdquo in Analysis of Marine Ecosystems LongburstEd Academic Press London UK 1981

[74] P A Meyers ldquoPolyunsaturated fatty acids in coral indicatorsof nutritional sourcesrdquoMarine Biology Letter vol 1 pp 69ndash751979

[75] J A Reiffel and A McDonald ldquoAntiarrhythmic effects ofomega-3 fatty acidsrdquo American Journal of Cardiology vol 98no 4 pp 50ndash60 2006

[76] P M Kris-Etherton A E Griel T L Psota S K Gebauer JZhang and T D Etherton ldquoDietary stearic acid and risk of car-diovascular disease intake sources digestion and absorptionrdquoLipids vol 40 no 12 pp 1193ndash1200 2005

[77] R P Mensink ldquoEffects of stearic acid on plasma lipid andlipoproteins in humansrdquo Lipids vol 40 no 12 Article ID L9817pp 1201ndash1205 2005

[78] B F Haumann ldquoStearic acid a different saturated fatty acidrdquoInform vol 9 no 3 pp 202ndash208 1998

[79] S M Grundy ldquoInfluence of stearic acid on cholesterolmetabolism relative to other long-chain fatty acidsrdquo AmericanJournal of Clinical Nutrition vol 60 no 6 pp 986ndash990 1994

[80] T A Pearson ldquoStearic acid a unique saturated fatty acidrdquo TheAmerican Journal of Clinical Nutrition vol 60 pp 983ndash10721994

[81] Z-R Zou Y-H Yi H-M Wu J-H Wu C-C Liaw and K-H Lee ldquoIntercedensides A-C three new cytotoxic triterpeneglycosides from the sea cucumber Mensamaria intercedensLampertrdquo Journal of Natural Products vol 66 no 8 pp 1055ndash1060 2003


[82] K Yamada A Hamada F Kisa T Miyamoto and R HiguchildquoConstituents of Holothuroidea 13 Structure of neuritogenicactive ganglioside molecular species from the sea cucumberStichopus chloronotusrdquo Chemical amp Pharmaceutical Bulletinvol 51 no 1 pp 46ndash52 2003

[83] J A Beutler T C McKee R W Fuller et al ldquoFrequentoccurrence of HIV-inhibitory sulphated polysaccharides inmarine invertebratesrdquo Antiviral Chemistry amp Chemotherapyvol 4 no 3 pp 167ndash172 1993

[84] CHuihui Y Ping andL Jianrong ldquoThepreparation of collagenpolypeptide with free radical scavenging ability purified fromAcaudina molpadioides Semperrdquo Journal of Chinese Institute ofFood Science and Technology vol 2010-01 2010

[85] Z Su X Zhou S H Loukin W J Haynes Y Saimi andC Kung ldquoThe use of yeast to understand TRP-channelmechanosensitivityrdquo Pflugers Archiv - European Journal ofPhysiology vol 458 no 5 pp 861ndash867 2009

[86] B D Fredalina B H Ridzwan A A Z Abidin et al ldquoFatty acidcompositions in local sea cucumber Stichopus chloronotus forwound healingrdquo General Pharmacology The Vascular Systemvol 33 no 4 pp 337ndash340 1999

[87] A C Rustan and A D Christian ldquoFatty acids structures andpropertiesrdquo in Encyclopedia of Life Sciences JohnWiley amp Sons2005

[88] J CDrazen C F PhlegerMAGuest andPDNichols ldquoLipidsterols and fatty acid composition of abyssal holothuriansand ophiuroids from the North-East Pacific Ocean food webimplicationsrdquo Comparative Biochemistry and PhysiologymdashPartB Biochemistry amp Molecular Biology vol 151 no 1 pp 79ndash872008

[89] A Gil ldquoPolyunsaturated fatty acids and inflammatory diseasesrdquoBiomedicine amp Pharmacotherapy vol 56 no 8 pp 388ndash3962002

[90] A M M Jais R McCulloch and K Croft ldquoFatty acid andamino acid composition in haruan as a potential role in woundhealingrdquo General Pharmacology The Vascular System vol 25no 5 pp 947ndash950 1994

Medicinal ChemistryInternational Journal of

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MEDIATORSINFLAMMATION

of

Submit your manuscripts atwwwhindawicom


medication and aphrodisiac dating back many centuries [4]To highlight the historical importance of these organisms inthe Far East [5] opined that sea cucumbers have been usefulin folk medicine where data in the literature suggests thatabout fifty-two species are commercially exploited as foodand most of these are native to the tropics and subtropics [6]

In addition to consumption as functional food resultsof published studies also suggest that various organs of seacucumbers contain numerous bioactives that show biologicalactivity which may have potential as therapeutic agentsin human medicine and for veterinary applications Someimportant compounds already identified in these marinespecies include sulfated polysaccharides such as fucosylatedchondroitin sulfate [7] sterols sterol glycosides frondogeninand its glycosides [8 9] Furthermore lectins heparincerebrosides gangliosides omega-6 and omega-3 fatty acids(n-6 and n-3 FA) that are known to exhibit multiple biologicalactivities in living cells are also among the substances isolatedfrom sea cucumbers These compounds are now in use inanticancer antiangiogenic anticoagulant antihypertensiveanti-inflammatory antimicrobial antifungal antioxidantantithrombotic and antitumor applications [8]

Among the species of sea cucumber so far evaluatedHolothuria leucospilota is particularly widely distributed inthe shallow reefs of tropical subtropical and Indo-Pacificregions including the Red Sea [10ndash12] They are also foundliving in the Pacific Ocean from Northern Australia south-wards to LordHowe and have been discovered in the tropicalwaters of the Indo-West Pacific feeding on phytoplanktonmicro- and macroalgae and bacteria [13] Analysis of Hleucospilota has revealed some unique additional bioactivecompounds that are of therapeutic significance includingphenolic compounds [14] essential and free fatty acids [1516] gangliosides [17] and carotenoids [18] among manyothers In other words the detection of these compounds inthe species has been considered as strong evidence to supportthe use and application of extracts from H Leucospilota infolklore medicine to treat several infections and diseases

Despite this relative advantage majority of studiesencountered on the species in the literature are primarilyfocused on its content of triterpene glycosides or some otherspecific compounds of interest to researchers The completeanalysis of bioactives in the species is yet to be carried outsuggesting that more quantitative research is required toevaluate the entire body organs ofH leucospilota for bioactivecomponents The present study was therefore aimed at fullyextracting and characterizing the complete bioactive com-ponents in the body organs of adult H leucospilota sampledfrom the coastal shores ofMalaysia in order to determine themetabolically and biologically active substances and the fattyacid (FA) constituents

2 Materials and Methods

21 Source and Preparation of Sea Cucumber Samples Seacucumbers used in the present study were sampled fromTeluk Kemang near Port Dickson (2∘ 271015840 466110158401015840N 101∘501015840 429810158401015840E) Malaysia Live adult samples were collectedby hand from the sea shores in plastic sample containers

during low tides Samples were washed free of debris withclean sea water kept in transparent polyethylene bags inchilled sampling boxes and transported to the laboratoryfacilities of Marine Biotechnology Institute of Biosciences(IBS) Universiti Putra Malaysia (UPM) Serdang SelangorMalaysia

In the laboratory liveH leucospilota samples were furthercleaned of sand salt water and left-over debris with distilledwater and then cut into small pieces (2 cm times 2 cm) withsterile knives forceps and other dissecting tools The bodywall Cuvierian tubules and visceral organs were separatedwashed and kept in separate sample bottles Separated organsamples were then lyophilized using a bench top K-manifoldfreeze-dryer (FreeZone Labconco USA) for 120 hours Thefreeze-dried samples were then ground with a blender (MagicBullet Model MBR-1101G USA) to fine powder quantifiedon electronic weighing balance (Model B12-FAJA Taiwan)and kept in dry sample bottles The dry powdered samples inbottles were covered with aluminum foil papers and storedat -20∘C until used during subsequent analyses within threeweeksThe powdered samples were appropriately labeled andidentified as body wall (BW) Cuvierian tubules (CT) andvisceral organs (VO) in subsequent evaluations

22 Crude Sample Extraction Procedure Extraction of bioac-tive constituents was performed ondry powderedH leucospi-lota organ samples using Soxhlet and conventional solventextraction techniques Briefly during the Soxhlet extractionknown volumes (vv label) of analytical grade solventsmethanolacetone (31 MA) isopropanolacetone (31 PA)ethanoldistilled water (31 ED) methanoldistilled water(31 MD) and iso-propanoldistilled water (31 PD) wereused Repeated extraction was carried out on each separatelyweighed sample per solvent mixture to extract bioactiveconstituents from various powdered sea cucumber organsamples Each sample extraction procedure lasted about 5hours Subsequently fractions from each repeated solventextraction were pooled together and filtered with Whatman(No 1) filter paper and the filtrate was evaporated to drynesson a rotary evaporator

In the conventional solvent extraction procedure thepowdered organ samples were weighed and dissolved in ana-lytical grade (vv label) acetone (A) and methanolacetone(11 MA) Approximately 200 g of BW 70 g CT and 73 gVO were extracted in 2 L 700 mL and 730 mL of extractionsolvent respectively (representing a concentration of 1 g oforgan sample in 10 mL of extraction solvent) Extractionwas carried out under ambient temperature for 4 days withconstant shaking according to slight modification to themethod described by [20] All extractions were carried outunder aseptic conditions in clean dry beakersThe procedurewas repeated many times (between twelve to fifteen cycles)until no further color changes were observed in the solventsBatches of the extracts for each sample were then pooledand filtered and the filtrate was concentrated on a rotaryevaporator as previously described Water (moisture) wasremoved from the extracts by freeze-drying and the dry organextracts (BW CT and VO) were then used in subsequentevaluations immediately or stored at -20∘C until used






C = (C times V)m

(1)




C = (C times V)m

(2)



C = (C times V)m

(3)






Phenols + + + + + + + + + +

Terpenes + + + + + + + + + +

Carbohydrates + + + + + + + + + +


Steroids - - + - + + + + +

Saponins - + - + - + + + + +


Glycosides - + - + - + + + + +

Flavonoids - - - + + + + + + +




3 Results











005

115

225

335

445

5

Conc

entr

atio

n (m

gg)









Table3Bioactivec

ompo

nentsd


C-MSindifferent

solventextractso

fbod

yorgans

ofseac

ucum

berHleucospilota

sampled

from

Peninsular

Malaysia

RTCom

poun

dsof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

EDBW

MDBW

PDBW

Ketones

5558


-Pentano

ne

3715

18869

27189

25

177

308

341

5533

4-methyl-3

-Penten-2-on

e05

382

18117

19-

--

-12

2Ph

enols

20225

24-bis(11-dim

ethylethyl)-Ph

enol-

27

111

213

359

214

-095

141

173

Alcohols

13367

2-Fu

ranm

ethano

l37

105

47

053

15042

-018

067

072

20783


38

--

--

--

--

-21017

10-M

ethyl-8


66

--

--

--

--

-226


l-

915

--

--

--

-018

2035

Glycerin

--

073

05

--

097

049

654

-Hydrocarbons

564

23-Ch

lorooctane

58

337

20413

20475

-12

-367

5808

2-Ch

lorooctane

68

105

95226

1145

-366

728

836

8933


25

402

39

-1

114

--

--

9075

3-ethyloctane

-545

--

--

--

--

5592

1-methyl-3-ethylC

yclopentane

-117

--

--

--

--

5592

Cyclop

entane1-ethyl-2-m

ethyl

--

--

519

--

--

-76

921-N

onene

11117

-059

08

--

--

-Fatty

acids

23117

Tetradecanoica

cid

--

2021

12-

116

104

093

065

25783

n-Hexadecanoica

cid

08

052

88

112

613

979

3127

568

343

26217

Z-11-Hexadecanoica

cid

--

57

-41

--

--

-26267

9-Hexadecenoica

cid

--

35

-27

079

48

324

241

167

29683

cis-9-O

ctadecenoica

cid(oleicacid)

--

045

077

05

-294

152

--

2453

3Eicosano

icacid

(arachidicacid)

--

017

051

--

132

068

022

-2713

3Heptadecano

icacid

--

--

05

--

172

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acidscontin

ued

10642

Cycloh

exaneb

utanoica

cid

-10

6-

--

--

--

-289

Octadecanoica

cid

--

1602

19057

354

372

133

085

Carboxylicacid

1065

Aceticacid

08

--

-08

036

-082

11097

14717

Croton

icacid

(2-butenoica

cid)

04

022

052

067

046

--

-016

04

Aldehydes

15267

24-

dimethylb

enzaldehyde

--

13083

26

085

-044

091

-14225

37-dimethyl-2

6-O

ctadienal

--

--

-464

--

--

1035

Furfu

ral

--

--

18083

--

--

22033

(Z)-9-

Octadecenal

--

-111

13-

--

--

Sterols

409

Stigmast-5

-en-3-ol(3120573

24120590

)-(sito

sterol)

--

05

-254

--

-215

47017

14-M

ethyl-e

rgost-8

-en-ol(3120573

5120572)-

--

--

--

077

734

--

38558


--

-085

--

-17

2319

Ergosta


512057222120576)-

--

--

--

--

103

-309


-10

8-

-36

316

--

337

356

33858

Cholesta-35-diene

-095

-043

08

263

--

229

196

37717

Ergosta

-422-diene

--

--

-19

8-

--

-35592

Ergosta

-4622-triene

--

--

04

--

--

121

Fatty


19592

9-Hexadecenoica

cidmethyleste

r-

336

032

128

13361

--

--

19308

Hexadecanoica

cidmethyleste

r-

786

-1763

-90

5024

--

-21225

Octadecanoica

cidmethyleste

r06

741

044

835

11482

--

025

033

21483

13-O

ctadecenoica

cidmethyleste

r-

196

-38

-17

2-

--

-2315

7-Hexadecenoica

cidmethyleste

r-

391

-212

-212

-013

-017

17783

Pentadecanoica

cidmethyleste

r-

033

-407

-17

2-

--

-19575

Hexadecenoica

cidmethyleste

r-

--

--

635

--

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acid


22292

Eicosano

icacid

methyleste

r-

17-

--

107

--

--

34875

11-Eicoseno

icacid

methyleste

r-

044

02

075

-014

217

121

607

359

17233

Methyltetradecano

ate

09

087

-337

-12

1-

--

-39717


cidethyleste

r(arachido

nica

cid)

-827

-105

11127

206

119

604

302

40725

581114

17-E

icosapentaenoica

cidmethyleste

r(EP

A)

-255

-557

-368

--

--

42842

47101316

1719

-Docosahexaeno

icacidm

ethyleste

r(DHA)

--

--

--

274

--

-Esters

31783


4-

-28

--

--

-114

24883


--

38

--

--

--

-17075


enoica

cid

-91

--

--

--

--

Amines

16017

2-methyl-2

-Propanamine

--

31

085

--

--

--

16308

244-Trim

ethyl-2

-Pentanamine

--

--

28

--

--

--representsn

otdetectableA

CTaceton

eCuvieria

ntubu

leM

ACTmethano

lacetone

Cuvieriantubu

leA

VOacetone

visceralorganMAV

Om

ethano

lacetone


body

wallM

ABW

methano

lacetone

body

tissuePA

BWisoprop

anolacetone

body

tissueED

BWethanoldistilled

waterbo

dywallMDBW

methano

ldistilled

waterbo

dywallPD

BWisoprop

anoldistilled

waterbo

dywallextracts


0

FAME

TPEALD

PNLCHO

ALCCBA ST FA AM

HCN KE

Compound group

5

10

15

20

25

30

35

Perc

enta

ge (

) in

extr

act













4 Discussion


























5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of







C = (C times V)m

(1)




C = (C times V)m

(2)



C = (C times V)m

(3)






Phenols + + + + + + + + + +

Terpenes + + + + + + + + + +

Carbohydrates + + + + + + + + + +


Steroids - - + - + + + + +

Saponins - + - + - + + + + +


Glycosides - + - + - + + + + +

Flavonoids - - - + + + + + + +




3 Results











005

115

225

335

445

5

Conc

entr

atio

n (m

gg)









Table3Bioactivec

ompo

nentsd


C-MSindifferent

solventextractso

fbod

yorgans

ofseac

ucum

berHleucospilota

sampled

from

Peninsular

Malaysia

RTCom

poun

dsof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

EDBW

MDBW

PDBW

Ketones

5558


-Pentano

ne

3715

18869

27189

25

177

308

341

5533

4-methyl-3

-Penten-2-on

e05

382

18117

19-

--

-12

2Ph

enols

20225

24-bis(11-dim

ethylethyl)-Ph

enol-

27

111

213

359

214

-095

141

173

Alcohols

13367

2-Fu

ranm

ethano

l37

105

47

053

15042

-018

067

072

20783


38

--

--

--

--

-21017

10-M

ethyl-8


66

--

--

--

--

-226


l-

915

--

--

--

-018

2035

Glycerin

--

073

05

--

097

049

654

-Hydrocarbons

564

23-Ch

lorooctane

58

337

20413

20475

-12

-367

5808

2-Ch

lorooctane

68

105

95226

1145

-366

728

836

8933


25

402

39

-1

114

--

--

9075

3-ethyloctane

-545

--

--

--

--

5592

1-methyl-3-ethylC

yclopentane

-117

--

--

--

--

5592

Cyclop

entane1-ethyl-2-m

ethyl

--

--

519

--

--

-76

921-N

onene

11117

-059

08

--

--

-Fatty

acids

23117

Tetradecanoica

cid

--

2021

12-

116

104

093

065

25783

n-Hexadecanoica

cid

08

052

88

112

613

979

3127

568

343

26217

Z-11-Hexadecanoica

cid

--

57

-41

--

--

-26267

9-Hexadecenoica

cid

--

35

-27

079

48

324

241

167

29683

cis-9-O

ctadecenoica

cid(oleicacid)

--

045

077

05

-294

152

--

2453

3Eicosano

icacid

(arachidicacid)

--

017

051

--

132

068

022

-2713

3Heptadecano

icacid

--

--

05

--

172

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acidscontin

ued

10642

Cycloh

exaneb

utanoica

cid

-10

6-

--

--

--

-289

Octadecanoica

cid

--

1602

19057

354

372

133

085

Carboxylicacid

1065

Aceticacid

08

--

-08

036

-082

11097

14717

Croton

icacid

(2-butenoica

cid)

04

022

052

067

046

--

-016

04

Aldehydes

15267

24-

dimethylb

enzaldehyde

--

13083

26

085

-044

091

-14225

37-dimethyl-2

6-O

ctadienal

--

--

-464

--

--

1035

Furfu

ral

--

--

18083

--

--

22033

(Z)-9-

Octadecenal

--

-111

13-

--

--

Sterols

409

Stigmast-5

-en-3-ol(3120573

24120590

)-(sito

sterol)

--

05

-254

--

-215

47017

14-M

ethyl-e

rgost-8

-en-ol(3120573

5120572)-

--

--

--

077

734

--

38558


--

-085

--

-17

2319

Ergosta


512057222120576)-

--

--

--

--

103

-309


-10

8-

-36

316

--

337

356

33858

Cholesta-35-diene

-095

-043

08

263

--

229

196

37717

Ergosta

-422-diene

--

--

-19

8-

--

-35592

Ergosta

-4622-triene

--

--

04

--

--

121

Fatty


19592

9-Hexadecenoica

cidmethyleste

r-

336

032

128

13361

--

--

19308

Hexadecanoica

cidmethyleste

r-

786

-1763

-90

5024

--

-21225

Octadecanoica

cidmethyleste

r06

741

044

835

11482

--

025

033

21483

13-O

ctadecenoica

cidmethyleste

r-

196

-38

-17

2-

--

-2315

7-Hexadecenoica

cidmethyleste

r-

391

-212

-212

-013

-017

17783

Pentadecanoica

cidmethyleste

r-

033

-407

-17

2-

--

-19575

Hexadecenoica

cidmethyleste

r-

--

--

635

--

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acid


22292

Eicosano

icacid

methyleste

r-

17-

--

107

--

--

34875

11-Eicoseno

icacid

methyleste

r-

044

02

075

-014

217

121

607

359

17233

Methyltetradecano

ate

09

087

-337

-12

1-

--

-39717


cidethyleste

r(arachido

nica

cid)

-827

-105

11127

206

119

604

302

40725

581114

17-E

icosapentaenoica

cidmethyleste

r(EP

A)

-255

-557

-368

--

--

42842

47101316

1719

-Docosahexaeno

icacidm

ethyleste

r(DHA)

--

--

--

274

--

-Esters

31783


4-

-28

--

--

-114

24883


--

38

--

--

--

-17075


enoica

cid

-91

--

--

--

--

Amines

16017

2-methyl-2

-Propanamine

--

31

085

--

--

--

16308

244-Trim

ethyl-2

-Pentanamine

--

--

28

--

--

--representsn

otdetectableA

CTaceton

eCuvieria

ntubu

leM

ACTmethano

lacetone

Cuvieriantubu

leA

VOacetone

visceralorganMAV

Om

ethano

lacetone


body

wallM

ABW

methano

lacetone

body

tissuePA

BWisoprop

anolacetone

body

tissueED

BWethanoldistilled

waterbo

dywallMDBW

methano

ldistilled

waterbo

dywallPD

BWisoprop

anoldistilled

waterbo

dywallextracts


0

FAME

TPEALD

PNLCHO

ALCCBA ST FA AM

HCN KE

Compound group

5

10

15

20

25

30

35

Perc

enta

ge (

) in

extr

act













4 Discussion


























5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of





Phenols + + + + + + + + + +

Terpenes + + + + + + + + + +

Carbohydrates + + + + + + + + + +


Steroids - - + - + + + + +

Saponins - + - + - + + + + +


Glycosides - + - + - + + + + +

Flavonoids - - - + + + + + + +




3 Results











005

115

225

335

445

5

Conc

entr

atio

n (m

gg)









Table3Bioactivec

ompo

nentsd


C-MSindifferent

solventextractso

fbod

yorgans

ofseac

ucum

berHleucospilota

sampled

from

Peninsular

Malaysia

RTCom

poun

dsof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

EDBW

MDBW

PDBW

Ketones

5558


-Pentano

ne

3715

18869

27189

25

177

308

341

5533

4-methyl-3

-Penten-2-on

e05

382

18117

19-

--

-12

2Ph

enols

20225

24-bis(11-dim

ethylethyl)-Ph

enol-

27

111

213

359

214

-095

141

173

Alcohols

13367

2-Fu

ranm

ethano

l37

105

47

053

15042

-018

067

072

20783


38

--

--

--

--

-21017

10-M

ethyl-8


66

--

--

--

--

-226


l-

915

--

--

--

-018

2035

Glycerin

--

073

05

--

097

049

654

-Hydrocarbons

564

23-Ch

lorooctane

58

337

20413

20475

-12

-367

5808

2-Ch

lorooctane

68

105

95226

1145

-366

728

836

8933


25

402

39

-1

114

--

--

9075

3-ethyloctane

-545

--

--

--

--

5592

1-methyl-3-ethylC

yclopentane

-117

--

--

--

--

5592

Cyclop

entane1-ethyl-2-m

ethyl

--

--

519

--

--

-76

921-N

onene

11117

-059

08

--

--

-Fatty

acids

23117

Tetradecanoica

cid

--

2021

12-

116

104

093

065

25783

n-Hexadecanoica

cid

08

052

88

112

613

979

3127

568

343

26217

Z-11-Hexadecanoica

cid

--

57

-41

--

--

-26267

9-Hexadecenoica

cid

--

35

-27

079

48

324

241

167

29683

cis-9-O

ctadecenoica

cid(oleicacid)

--

045

077

05

-294

152

--

2453

3Eicosano

icacid

(arachidicacid)

--

017

051

--

132

068

022

-2713

3Heptadecano

icacid

--

--

05

--

172

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acidscontin

ued

10642

Cycloh

exaneb

utanoica

cid

-10

6-

--

--

--

-289

Octadecanoica

cid

--

1602

19057

354

372

133

085

Carboxylicacid

1065

Aceticacid

08

--

-08

036

-082

11097

14717

Croton

icacid

(2-butenoica

cid)

04

022

052

067

046

--

-016

04

Aldehydes

15267

24-

dimethylb

enzaldehyde

--

13083

26

085

-044

091

-14225

37-dimethyl-2

6-O

ctadienal

--

--

-464

--

--

1035

Furfu

ral

--

--

18083

--

--

22033

(Z)-9-

Octadecenal

--

-111

13-

--

--

Sterols

409

Stigmast-5

-en-3-ol(3120573

24120590

)-(sito

sterol)

--

05

-254

--

-215

47017

14-M

ethyl-e

rgost-8

-en-ol(3120573

5120572)-

--

--

--

077

734

--

38558


--

-085

--

-17

2319

Ergosta


512057222120576)-

--

--

--

--

103

-309


-10

8-

-36

316

--

337

356

33858

Cholesta-35-diene

-095

-043

08

263

--

229

196

37717

Ergosta

-422-diene

--

--

-19

8-

--

-35592

Ergosta

-4622-triene

--

--

04

--

--

121

Fatty


19592

9-Hexadecenoica

cidmethyleste

r-

336

032

128

13361

--

--

19308

Hexadecanoica

cidmethyleste

r-

786

-1763

-90

5024

--

-21225

Octadecanoica

cidmethyleste

r06

741

044

835

11482

--

025

033

21483

13-O

ctadecenoica

cidmethyleste

r-

196

-38

-17

2-

--

-2315

7-Hexadecenoica

cidmethyleste

r-

391

-212

-212

-013

-017

17783

Pentadecanoica

cidmethyleste

r-

033

-407

-17

2-

--

-19575

Hexadecenoica

cidmethyleste

r-

--

--

635

--

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acid


22292

Eicosano

icacid

methyleste

r-

17-

--

107

--

--

34875

11-Eicoseno

icacid

methyleste

r-

044

02

075

-014

217

121

607

359

17233

Methyltetradecano

ate

09

087

-337

-12

1-

--

-39717


cidethyleste

r(arachido

nica

cid)

-827

-105

11127

206

119

604

302

40725

581114

17-E

icosapentaenoica

cidmethyleste

r(EP

A)

-255

-557

-368

--

--

42842

47101316

1719

-Docosahexaeno

icacidm

ethyleste

r(DHA)

--

--

--

274

--

-Esters

31783


4-

-28

--

--

-114

24883


--

38

--

--

--

-17075


enoica

cid

-91

--

--

--

--

Amines

16017

2-methyl-2

-Propanamine

--

31

085

--

--

--

16308

244-Trim

ethyl-2

-Pentanamine

--

--

28

--

--

--representsn

otdetectableA

CTaceton

eCuvieria

ntubu

leM

ACTmethano

lacetone

Cuvieriantubu

leA

VOacetone

visceralorganMAV

Om

ethano

lacetone


body

wallM

ABW

methano

lacetone

body

tissuePA

BWisoprop

anolacetone

body

tissueED

BWethanoldistilled

waterbo

dywallMDBW

methano

ldistilled

waterbo

dywallPD

BWisoprop

anoldistilled

waterbo

dywallextracts


0

FAME

TPEALD

PNLCHO

ALCCBA ST FA AM

HCN KE

Compound group

5

10

15

20

25

30

35

Perc

enta

ge (

) in

extr

act













4 Discussion


























5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of








005

115

225

335

445

5

Conc

entr

atio

n (m

gg)









Table3Bioactivec

ompo

nentsd


C-MSindifferent

solventextractso

fbod

yorgans

ofseac

ucum

berHleucospilota

sampled

from

Peninsular

Malaysia

RTCom

poun

dsof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

EDBW

MDBW

PDBW

Ketones

5558


-Pentano

ne

3715

18869

27189

25

177

308

341

5533

4-methyl-3

-Penten-2-on

e05

382

18117

19-

--

-12

2Ph

enols

20225

24-bis(11-dim

ethylethyl)-Ph

enol-

27

111

213

359

214

-095

141

173

Alcohols

13367

2-Fu

ranm

ethano

l37

105

47

053

15042

-018

067

072

20783


38

--

--

--

--

-21017

10-M

ethyl-8


66

--

--

--

--

-226


l-

915

--

--

--

-018

2035

Glycerin

--

073

05

--

097

049

654

-Hydrocarbons

564

23-Ch

lorooctane

58

337

20413

20475

-12

-367

5808

2-Ch

lorooctane

68

105

95226

1145

-366

728

836

8933


25

402

39

-1

114

--

--

9075

3-ethyloctane

-545

--

--

--

--

5592

1-methyl-3-ethylC

yclopentane

-117

--

--

--

--

5592

Cyclop

entane1-ethyl-2-m

ethyl

--

--

519

--

--

-76

921-N

onene

11117

-059

08

--

--

-Fatty

acids

23117

Tetradecanoica

cid

--

2021

12-

116

104

093

065

25783

n-Hexadecanoica

cid

08

052

88

112

613

979

3127

568

343

26217

Z-11-Hexadecanoica

cid

--

57

-41

--

--

-26267

9-Hexadecenoica

cid

--

35

-27

079

48

324

241

167

29683

cis-9-O

ctadecenoica

cid(oleicacid)

--

045

077

05

-294

152

--

2453

3Eicosano

icacid

(arachidicacid)

--

017

051

--

132

068

022

-2713

3Heptadecano

icacid

--

--

05

--

172

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acidscontin

ued

10642

Cycloh

exaneb

utanoica

cid

-10

6-

--

--

--

-289

Octadecanoica

cid

--

1602

19057

354

372

133

085

Carboxylicacid

1065

Aceticacid

08

--

-08

036

-082

11097

14717

Croton

icacid

(2-butenoica

cid)

04

022

052

067

046

--

-016

04

Aldehydes

15267

24-

dimethylb

enzaldehyde

--

13083

26

085

-044

091

-14225

37-dimethyl-2

6-O

ctadienal

--

--

-464

--

--

1035

Furfu

ral

--

--

18083

--

--

22033

(Z)-9-

Octadecenal

--

-111

13-

--

--

Sterols

409

Stigmast-5

-en-3-ol(3120573

24120590

)-(sito

sterol)

--

05

-254

--

-215

47017

14-M

ethyl-e

rgost-8

-en-ol(3120573

5120572)-

--

--

--

077

734

--

38558


--

-085

--

-17

2319

Ergosta


512057222120576)-

--

--

--

--

103

-309


-10

8-

-36

316

--

337

356

33858

Cholesta-35-diene

-095

-043

08

263

--

229

196

37717

Ergosta

-422-diene

--

--

-19

8-

--

-35592

Ergosta

-4622-triene

--

--

04

--

--

121

Fatty


19592

9-Hexadecenoica

cidmethyleste

r-

336

032

128

13361

--

--

19308

Hexadecanoica

cidmethyleste

r-

786

-1763

-90

5024

--

-21225

Octadecanoica

cidmethyleste

r06

741

044

835

11482

--

025

033

21483

13-O

ctadecenoica

cidmethyleste

r-

196

-38

-17

2-

--

-2315

7-Hexadecenoica

cidmethyleste

r-

391

-212

-212

-013

-017

17783

Pentadecanoica

cidmethyleste

r-

033

-407

-17

2-

--

-19575

Hexadecenoica

cidmethyleste

r-

--

--

635

--

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acid


22292

Eicosano

icacid

methyleste

r-

17-

--

107

--

--

34875

11-Eicoseno

icacid

methyleste

r-

044

02

075

-014

217

121

607

359

17233

Methyltetradecano

ate

09

087

-337

-12

1-

--

-39717


cidethyleste

r(arachido

nica

cid)

-827

-105

11127

206

119

604

302

40725

581114

17-E

icosapentaenoica

cidmethyleste

r(EP

A)

-255

-557

-368

--

--

42842

47101316

1719

-Docosahexaeno

icacidm

ethyleste

r(DHA)

--

--

--

274

--

-Esters

31783


4-

-28

--

--

-114

24883


--

38

--

--

--

-17075


enoica

cid

-91

--

--

--

--

Amines

16017

2-methyl-2

-Propanamine

--

31

085

--

--

--

16308

244-Trim

ethyl-2

-Pentanamine

--

--

28

--

--

--representsn

otdetectableA

CTaceton

eCuvieria

ntubu

leM

ACTmethano

lacetone

Cuvieriantubu

leA

VOacetone

visceralorganMAV

Om

ethano

lacetone


body

wallM

ABW

methano

lacetone

body

tissuePA

BWisoprop

anolacetone

body

tissueED

BWethanoldistilled

waterbo

dywallMDBW

methano

ldistilled

waterbo

dywallPD

BWisoprop

anoldistilled

waterbo

dywallextracts


0

FAME

TPEALD

PNLCHO

ALCCBA ST FA AM

HCN KE

Compound group

5

10

15

20

25

30

35

Perc

enta

ge (

) in

extr

act













4 Discussion


























5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



Table3Bioactivec

ompo

nentsd


C-MSindifferent

solventextractso

fbod

yorgans

ofseac

ucum

berHleucospilota

sampled

from

Peninsular

Malaysia

RTCom

poun

dsof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

EDBW

MDBW

PDBW

Ketones

5558


-Pentano

ne

3715

18869

27189

25

177

308

341

5533

4-methyl-3

-Penten-2-on

e05

382

18117

19-

--

-12

2Ph

enols

20225

24-bis(11-dim

ethylethyl)-Ph

enol-

27

111

213

359

214

-095

141

173

Alcohols

13367

2-Fu

ranm

ethano

l37

105

47

053

15042

-018

067

072

20783


38

--

--

--

--

-21017

10-M

ethyl-8


66

--

--

--

--

-226


l-

915

--

--

--

-018

2035

Glycerin

--

073

05

--

097

049

654

-Hydrocarbons

564

23-Ch

lorooctane

58

337

20413

20475

-12

-367

5808

2-Ch

lorooctane

68

105

95226

1145

-366

728

836

8933


25

402

39

-1

114

--

--

9075

3-ethyloctane

-545

--

--

--

--

5592

1-methyl-3-ethylC

yclopentane

-117

--

--

--

--

5592

Cyclop

entane1-ethyl-2-m

ethyl

--

--

519

--

--

-76

921-N

onene

11117

-059

08

--

--

-Fatty

acids

23117

Tetradecanoica

cid

--

2021

12-

116

104

093

065

25783

n-Hexadecanoica

cid

08

052

88

112

613

979

3127

568

343

26217

Z-11-Hexadecanoica

cid

--

57

-41

--

--

-26267

9-Hexadecenoica

cid

--

35

-27

079

48

324

241

167

29683

cis-9-O

ctadecenoica

cid(oleicacid)

--

045

077

05

-294

152

--

2453

3Eicosano

icacid

(arachidicacid)

--

017

051

--

132

068

022

-2713

3Heptadecano

icacid

--

--

05

--

172

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acidscontin

ued

10642

Cycloh

exaneb

utanoica

cid

-10

6-

--

--

--

-289

Octadecanoica

cid

--

1602

19057

354

372

133

085

Carboxylicacid

1065

Aceticacid

08

--

-08

036

-082

11097

14717

Croton

icacid

(2-butenoica

cid)

04

022

052

067

046

--

-016

04

Aldehydes

15267

24-

dimethylb

enzaldehyde

--

13083

26

085

-044

091

-14225

37-dimethyl-2

6-O

ctadienal

--

--

-464

--

--

1035

Furfu

ral

--

--

18083

--

--

22033

(Z)-9-

Octadecenal

--

-111

13-

--

--

Sterols

409

Stigmast-5

-en-3-ol(3120573

24120590

)-(sito

sterol)

--

05

-254

--

-215

47017

14-M

ethyl-e

rgost-8

-en-ol(3120573

5120572)-

--

--

--

077

734

--

38558


--

-085

--

-17

2319

Ergosta


512057222120576)-

--

--

--

--

103

-309


-10

8-

-36

316

--

337

356

33858

Cholesta-35-diene

-095

-043

08

263

--

229

196

37717

Ergosta

-422-diene

--

--

-19

8-

--

-35592

Ergosta

-4622-triene

--

--

04

--

--

121

Fatty


19592

9-Hexadecenoica

cidmethyleste

r-

336

032

128

13361

--

--

19308

Hexadecanoica

cidmethyleste

r-

786

-1763

-90

5024

--

-21225

Octadecanoica

cidmethyleste

r06

741

044

835

11482

--

025

033

21483

13-O

ctadecenoica

cidmethyleste

r-

196

-38

-17

2-

--

-2315

7-Hexadecenoica

cidmethyleste

r-

391

-212

-212

-013

-017

17783

Pentadecanoica

cidmethyleste

r-

033

-407

-17

2-

--

-19575

Hexadecenoica

cidmethyleste

r-

--

--

635

--

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acid


22292

Eicosano

icacid

methyleste

r-

17-

--

107

--

--

34875

11-Eicoseno

icacid

methyleste

r-

044

02

075

-014

217

121

607

359

17233

Methyltetradecano

ate

09

087

-337

-12

1-

--

-39717


cidethyleste

r(arachido

nica

cid)

-827

-105

11127

206

119

604

302

40725

581114

17-E

icosapentaenoica

cidmethyleste

r(EP

A)

-255

-557

-368

--

--

42842

47101316

1719

-Docosahexaeno

icacidm

ethyleste

r(DHA)

--

--

--

274

--

-Esters

31783


4-

-28

--

--

-114

24883


--

38

--

--

--

-17075


enoica

cid

-91

--

--

--

--

Amines

16017

2-methyl-2

-Propanamine

--

31

085

--

--

--

16308

244-Trim

ethyl-2

-Pentanamine

--

--

28

--

--

--representsn

otdetectableA

CTaceton

eCuvieria

ntubu

leM

ACTmethano

lacetone

Cuvieriantubu

leA

VOacetone

visceralorganMAV

Om

ethano

lacetone


body

wallM

ABW

methano

lacetone

body

tissuePA

BWisoprop

anolacetone

body

tissueED

BWethanoldistilled

waterbo

dywallMDBW

methano

ldistilled

waterbo

dywallPD

BWisoprop

anoldistilled

waterbo

dywallextracts


0

FAME

TPEALD

PNLCHO

ALCCBA ST FA AM

HCN KE

Compound group

5

10

15

20

25

30

35

Perc

enta

ge (

) in

extr

act













4 Discussion


























5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acidscontin

ued

10642

Cycloh

exaneb

utanoica

cid

-10

6-

--

--

--

-289

Octadecanoica

cid

--

1602

19057

354

372

133

085

Carboxylicacid

1065

Aceticacid

08

--

-08

036

-082

11097

14717

Croton

icacid

(2-butenoica

cid)

04

022

052

067

046

--

-016

04

Aldehydes

15267

24-

dimethylb

enzaldehyde

--

13083

26

085

-044

091

-14225

37-dimethyl-2

6-O

ctadienal

--

--

-464

--

--

1035

Furfu

ral

--

--

18083

--

--

22033

(Z)-9-

Octadecenal

--

-111

13-

--

--

Sterols

409

Stigmast-5

-en-3-ol(3120573

24120590

)-(sito

sterol)

--

05

-254

--

-215

47017

14-M

ethyl-e

rgost-8

-en-ol(3120573

5120572)-

--

--

--

077

734

--

38558


--

-085

--

-17

2319

Ergosta


512057222120576)-

--

--

--

--

103

-309


-10

8-

-36

316

--

337

356

33858

Cholesta-35-diene

-095

-043

08

263

--

229

196

37717

Ergosta

-422-diene

--

--

-19

8-

--

-35592

Ergosta

-4622-triene

--

--

04

--

--

121

Fatty


19592

9-Hexadecenoica

cidmethyleste

r-

336

032

128

13361

--

--

19308

Hexadecanoica

cidmethyleste

r-

786

-1763

-90

5024

--

-21225

Octadecanoica

cidmethyleste

r06

741

044

835

11482

--

025

033

21483

13-O

ctadecenoica

cidmethyleste

r-

196

-38

-17

2-

--

-2315

7-Hexadecenoica

cidmethyleste

r-

391

-212

-212

-013

-017

17783

Pentadecanoica

cidmethyleste

r-

033

-407

-17

2-

--

-19575

Hexadecenoica

cidmethyleste

r-

--

--

635

--

--


Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acid


22292

Eicosano

icacid

methyleste

r-

17-

--

107

--

--

34875

11-Eicoseno

icacid

methyleste

r-

044

02

075

-014

217

121

607

359

17233

Methyltetradecano

ate

09

087

-337

-12

1-

--

-39717


cidethyleste

r(arachido

nica

cid)

-827

-105

11127

206

119

604

302

40725

581114

17-E

icosapentaenoica

cidmethyleste

r(EP

A)

-255

-557

-368

--

--

42842

47101316

1719

-Docosahexaeno

icacidm

ethyleste

r(DHA)

--

--

--

274

--

-Esters

31783


4-

-28

--

--

-114

24883


--

38

--

--

--

-17075


enoica

cid

-91

--

--

--

--

Amines

16017

2-methyl-2

-Propanamine

--

31

085

--

--

--

16308

244-Trim

ethyl-2

-Pentanamine

--

--

28

--

--

--representsn

otdetectableA

CTaceton

eCuvieria

ntubu

leM

ACTmethano

lacetone

Cuvieriantubu

leA

VOacetone

visceralorganMAV

Om

ethano

lacetone


body

wallM

ABW

methano

lacetone

body

tissuePA

BWisoprop

anolacetone

body

tissueED

BWethanoldistilled

waterbo

dywallMDBW

methano

ldistilled

waterbo

dywallPD

BWisoprop

anoldistilled

waterbo

dywallextracts


0

FAME

TPEALD

PNLCHO

ALCCBA ST FA AM

HCN KE

Compound group

5

10

15

20

25

30

35

Perc

enta

ge (

) in

extr

act













4 Discussion


























5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



Table3Con

tinued

RTCom

poun

dof

Com

poun

dsin

extract

AC

TMAC

TAV

OMAV

OABW

MABW

PABW

PDBW

EDBW

MDBW

Fatty

acid


22292

Eicosano

icacid

methyleste

r-

17-

--

107

--

--

34875

11-Eicoseno

icacid

methyleste

r-

044

02

075

-014

217

121

607

359

17233

Methyltetradecano

ate

09

087

-337

-12

1-

--

-39717


cidethyleste

r(arachido

nica

cid)

-827

-105

11127

206

119

604

302

40725

581114

17-E

icosapentaenoica

cidmethyleste

r(EP

A)

-255

-557

-368

--

--

42842

47101316

1719

-Docosahexaeno

icacidm

ethyleste

r(DHA)

--

--

--

274

--

-Esters

31783


4-

-28

--

--

-114

24883


--

38

--

--

--

-17075


enoica

cid

-91

--

--

--

--

Amines

16017

2-methyl-2

-Propanamine

--

31

085

--

--

--

16308

244-Trim

ethyl-2

-Pentanamine

--

--

28

--

--

--representsn

otdetectableA

CTaceton

eCuvieria

ntubu

leM

ACTmethano

lacetone

Cuvieriantubu

leA

VOacetone

visceralorganMAV

Om

ethano

lacetone


body

wallM

ABW

methano

lacetone

body

tissuePA

BWisoprop

anolacetone

body

tissueED

BWethanoldistilled

waterbo

dywallMDBW

methano

ldistilled

waterbo

dywallPD

BWisoprop

anoldistilled

waterbo

dywallextracts


0

FAME

TPEALD

PNLCHO

ALCCBA ST FA AM

HCN KE

Compound group

5

10

15

20

25

30

35

Perc

enta

ge (

) in

extr

act













4 Discussion


























5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



0

FAME

TPEALD

PNLCHO

ALCCBA ST FA AM

HCN KE

Compound group

5

10

15

20

25

30

35

Perc

enta

ge (

) in

extr

act













4 Discussion


























5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of







4 Discussion


























5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of
























5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of














5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of





5 Conclusion


Data Availability




Acknowledgments



References




































































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of






















































































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of





















































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of


















Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of








Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of


Documents

Extraction and Characterization of Organ Components of the ...downloads.hindawi.com/journals/bmri/2019/2640684.pdf · ResearchArticle Extraction and Characterization of Organ Components